CLINICAL REPORT

A Novel Mutation in COL4A1 Gene: A Possible
Cause Of Early Postnatal Cerebrovascular Events
Alice Decio,1 Davide Tonduti,1 Anna Pichiecchio,2 Annalisa Vetro,3 Roberto Ciccone,4,5
Ivan Limongelli,4 Roberto Giorda,6 Lorella Caffi,7 Umberto Balottin,1,8 Orsetta Zuffardi,5
and Simona Orcesi8*
1

Child Neurology and Psychiatry Unit, Department of Brain and Behavioural Sciences, University of Pavia, Pavia, Italy

2

Department of Neuroradiology, C. Mondino National Neurological Institute, Pavia, Italy

3

Biotechnology Research Laboratory, IRCCS Policlinico San Matteo, Pavia, Italy
Center for Genetic and Therapeutic Approaches, C. Mondino National Neurological Institute, Pavia, Italy

4
5

Department of Molecular Medicine, University of Pavia, Pavia, Italy

6

Molecular Biology Laboratory, IRCCS Scientific Institute Eugenio Medea, Bosisio Parini, Italy
Child Neurology and Psychiatry Unit, Azienda Ospedaliera Papa Giovanni XXIII, Bergamo, Italy

7
8

Child Neurology and Psychiatry Unit, C. Mondino National Neurological Institute, Pavia, Italy

Manuscript Received: 22 May 2014; Manuscript Accepted: 14 November 2014

COL4A1 is located in humans on chromosome13q34 and it
encodes the alpha 1 chain of type IV collagen, a component of
basal membrane. It is expressed mainly in the brain, muscles,
kidneys and eyes. Different COL4A1 mutations have been
reported in many patients who present a very wide spectrum
of clinical symptoms. They typically show a multisystemic phenotype. Here we report on the case of a patient carrying a novel de
novo splicing mutation of COL4A1 associated with a distinctive
clinical picture characterized by onset in infancy and an unusual
evolution of the neuroradiological features. At three months of
age, the child was diagnosed with a congenital cataract, while his
brain MRI was normal. Over the following years, the patient
developed focal epilepsy, mild diplegia, asymptomatic microhematuria, raised creatine kinase levels, MRI white matter
abnormalities and brain calcification on CT. During the neuroradiological follow-up the extension and intensity of the brain
lesions progressively decreased. The significance of a second
variant in COL4A1 carried by the child and inherited from his
father remains to be clarified. In conclusion, our patient shows
new aspects of this collagenopathy and possibly a COL4A1
compound heterozygosity. Ó 2015 Wiley Periodicals, Inc.

Key words: COL4A1

gene; infants; leukoencephalopathy;

cerebral calcification

INTRODUCTION
COL4A1 encodes the alpha 1 chain of collagen type IV, a fundamental component of vascular basal membrane. The gene is
expressed mainly in the brain, muscles, kidneys and eyes.
Type IV collagen is made from six types of homologous chains
indicated as alpha1–6. These chains assemble as heterotrimers.

Ó 2015 Wiley Periodicals, Inc.

How to Cite this Article:
Decio A,Tonduti D, Pichiecchio A, Vetro
A, Ciccone R, Limongelli I, Giorda R, Caffi
L, Balottin U, Zuffardi O, Orcesi S. 2015. A
novel mutation in COL4A1 gene: A possible
cause of early postnatal cerebrovascular
events.
Am J Med Genet Part A. 9999:1–6.

Every tissue containing collagen type IV in its basal membrane
has a distinctive expression of alpha chains. Alpha chain heterotrimers associate in dimers and then in tetramers, forming the
complex collagen type IV suprastructure [Volonghi et al., 2010].
COL4A1 is located in humans on chromosome 13q34. Different
COL4A1 mutations have been reported in numerous patients who
present a very wide spectrum of clinical symptoms; no clear
genotype-phenotype association has been found [Gould et al.,
2005; Breedveld et al., 2006; Gould et al., 2006; Van der Knaap
Conflicts of interest: none.
Grant sponsor: Regione Lombardia government; Grant number: DGRS
13465–22/12/2010; Grant sponsor: PRIN 2010–2011; Grant number:
20108WT59Y_003.
�
Correspondence to:
Simona Orcesi, M.D., Child Neurology and Psychiatry Unit, C. Mondino
National Neurological Institute, via Mondino 2, 27100 Pavia, Italy.
E-mail: simona.orcesi@mondino.it
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2014
DOI 10.1002/ajmg.a.36907

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AMERICAN JOURNAL OF MEDICAL GENETICS PART A

et al., 2006; Sibon et al., 2007; Vahedi et al., 2007; Alamowitch et al.,
2009; Bilguvar et al., 2009; De Vries et al., 2009, Shah et al., 2010;
Livingston et al., 2011; Meuwissen et al., 2011; Tonduti et al., 2012;
Lemmens et al., 2013].
The presence of a multisystemic phenotype is one of the main
characteristics of COL4A1-mutated patients [Vahedi and
Alamowitch, 2011]. Typically the brain, eyes, kidneys and muscles
are variably involved in the disease [Tonduti et al., 2012]. The
presence of white matter involvement is typically linked to cerebral
hemorrhages and/or small vessel ischemic damage [Van der Knaap
et al., 2006; Corlobe et al., 2013]. A specific pattern of intracranial
calcification has been associated with mutations in the gene [Livingston et al., 2011; Tonduti et al., 2012].
Here we report on the case of a patient carrying a novel de novo
splicing mutation of COL4A1 associated with a distinctive clinical
picture characterized by onset in infancy and an unusual evolution
of the neuroradiological features.

CLINICAL REPORT
The child was born after an uneventful pregnancy. The delivery, too,
was uneventful and the child’s auxological parameters at birth were:
weight 3190 g (25–50th centile), length 48 cm (10–25th centile);
head circumference was not available. When the child was three
months old, his parents started to notice erratic, chaotic eye movements. Ophthalmological examination showed congenital bilateral
cataract, without other abnormalities. Brain magnetic resonance
imaging (MRI) at this time was normal (Fig. 1). A complete heart
assessment, abdominal ultrasound, metabolic examination for
plasma and urinary amino acids and urinary organic acids, and
screening for galactosemia were all normal. Karyotype analysis was
normal. Neurological examination showed microcephaly and nystagmus, otherwise it was normal. The child underwent surgical

implant of artificial lenses and started visual rehabilitation training.
The child’s motor and language development proceeded normally
until the age of two and a half years when he started to present focal
epileptic seizures with secondary generalization. An electroencephalogram showed epileptic slow waves in the right frontal hemisphere, spreading during sleep. The patient started antiepileptic
therapy, but oxcarbazepine failed to completely control the seizures
and focal episodes kept occurring with monthly frequency. On
neurological examination, the child showed mild, non-disabling
spastic diplegia with good cognitive performance.
A month and a half after the first seizure, a CT scan and a new
MRI were performed, which showed (Fig. 2A,B) bilateral confluent
periventricular and deep white matter areas of hypersignal intensity
(T2 and FLAIR sequences) and a left frontal periventricular cyst.
Hemosiderinic deposits were observed in the right frontoparietal
cortical-subcortical region (Fig. 2C) and CT scan revealed punctate
calcification in the frontal periventricular and basal ganglia areas.
At six years of age, the child presented a paroxysmal episode
characterized by painful cramps in his right arm subsequently also
involving his right leg. He had a creatine phosphokinase (CPK) level
of 5504 U\L (normal values 60–190), which increased to 26.342 U\L
the following day. Thereafter, CPK levels were monitored regularly
and they were always raised, reaching values higher than 2000 U\L.
The neurological examination was unchanged.
The child also presents asymptomatic microhematuria without
proteinuria. This has been evident since his first evaluation at the
age of 3 years.
During his follow-up, the boy underwent three additional MRI
evaluations (at 4, 6, and 8 years of age), which showed progressive
attenuation of the white matter areas of signal hyperintensity (Fig. 3).
Molecular analysis of the COL4A1 gene was performed using a
next-generation sequencing panel for small vessel diseases and
ischemic strokes on a Genome Analyzer IIx. The analysis identified

FIG. 1. Axial T2 MR images (A,B) performed at the age of 3 months, showing white matter myelination consistent with the child’s age.

DECIO ET AL.

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FIG. 2. T2-weighted axial MR images (A,B) and weighted Fast Field Echo (FFE) image (C) performed at the age of two and a half years. The T2
MR images (A,B) show bilateral mainly periventricular and deep white matter hyperintense alterations and a left frontal periventricular cyst.
Slight enlargement of the right lateral ventricle is also evident. The axial FFE image (C) shows a small punctate focus of susceptibility, likely
due to hemosiderin, in the right corticosubcortical rolandic parasagittal region.

two heterozygous variants affecting the gene, both confirmed by
Sanger sequencing (Fig. 4). The first one, c.2458 þ 1G>A, was
located at the donor splicing site of intron 31. Parental DNA
examination showed this novel mutation to be de novo. The second
mutation, c.3712C>T, affecting exon 42 of the gene and resulting in
a p.Arg1238Cys substitution, was reported in dbSNP138
(rs148801165) with a population frequency well below 1%. This
mutation was found to be inherited from the father. Both variants
were predicted to be “damaging” by at least one of the in silico
prediction tools used (Table I). In particular, the c.2458 þ 1G>A

mutation was predicted to cause skipping of exon 31 during
splicing. Real Time-PCR performed on mRNA from cultured
skin fibroblasts of the patient confirmed this hypothesis (Fig. 5).
Our patient’s father carried the c.3712C>T mutation. He is a
53-year-old man who has been affected by Raynaud’s phenomenon
and muscular cramps since he was young; he occasionally presents
slightly raised CPK values (highest value 312 U\L- normal values
60–190). A complete ophthalmological assessment revealed absence of retinal arterial tortuosities and completely normal fundus
oculi. Renal function was normal and renal and heart ultrasound

FIG. 3. (A,B,C) T2-weighted axial MRI performed when the patient was 8 years old, showing marked reduction of the previous bilateral
periventricular and deep white matter lesions, with persistence of the left frontal periventricular cyst.

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AMERICAN JOURNAL OF MEDICAL GENETICS PART A

FIG. 4. Detailed view of individual sequencing reads (horizontal gray bars) overlapping the COL4A1 mutations identified by NGS, as visualized
by Integrative Genomics Viewer (IGV) (Thorvaldsdóttir et al., 2013). A coverage plot (vertical gray bars) is displayed for each mutation. Reads
are aligned to the reference genome, whose sequence is shown below. The c.2458 þ 1G>A mutation is shown in panel (A) together with the
corresponding Sanger sequencing confirmation; (B) the inherited c.3712C>T mutation is shown in (C) with (D) its Sanger sequencing
confirmation. The medium coverage for the gene was above 200X and sequencing was performed using a 100bp paired-end protocol, as
reported elsewhere (Andolfo et al., 2013).

examination did not show abnormalities. Brain MRI did not show
lesions due to ischemic or hemorrhagic events, but revealed a
dermoid cyst in the superior cerebellar paravermian region. The
proband’s younger brother is 5 years old and presents asymptomatic microhematuria without proteinuria and moderate language
delay. A recent brain MRI was normal, revealing only a mildly
dysmorphic corpus callosum; no ischemic nor hemorrhagic lesions

were observed. Sanger sequencing showed neither of the two
mutations.

DISCUSSION
We report on the case of a patient carrying two variants in COL4A1.
One of them, a novel mutation, arose de novo in the proband and is

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TABLE I. Details of the Mutations Identified in Our Patient Including the Results of Three in Silico Prediction Tools.
Genomic position
chr13:110831269C>T
chr13:110822924G>A

HGVS label
NM_001845.4:c.2458þ1G>A
NM_001845.4:c.3712C>T

Inheritance
de novo
paternal

Mutation taster
disease causing
disease causing

SIFT
NA
tolerated

Polyphen2
NA
probably damaging

NA, not assessed because predictions are active only on missense mutations.

located at the donor splice site of intron 31 (c.2458 þ 1G>A). We
demonstrated that this variant, leading to the elimination of an
entire exon from the final transcript, causes a significant modification of protein structure and function.
This child’s clinical picture was characterized by mild spastic
diplegia with abnormal periventricular white matter. As previously reported in other COL4A1-mutated patients, signs of
renal, ocular and muscular involvement were evident [Vahedi

FIG. 5. Skipping of exon 31 in the COL4A1 transcript from
proband’s fibroblasts. (A) agarose gel analysis of the portion of the
COL4A1 transcript spanning exon 31 in the proband (P), two control
fibroblast samples (C1 and C2), ovary (O), and water control (N).
The shorter fragment amplified only in the proband is indicated by
an arrow. DNA Molecular Weight Marker V (Roche) was used as a
marker (M). (B) detail of the sequence of the short transcript from
the proband showing the junction between exons 30 and 32 and
skipping of exon 31. Total RNA was extracted with Trizol (Life
Technologies, Monza, Italy); cDNA synthesis was performed with
Ready-To-Go You-Prime First strand beads (Amersham, Milan, Italy)
and random hexamers; Non-quantitative RT-PCR of the portion of
COL4A1 spanning exon 31 was performed in 25 ml reactions, using
primers COL4A1-E29_30-F(50 -CTCCCTTGGAACCTGGAAAC-30 ) and
COL4A1-E32_33-R(50 -GCAGGGCCAGAAGGGAGAG-30 ) and JumpStart
Red ACCUTaq LA DNA polymerase (Sigma). PCR protocol: 1 min at
96 ˚C; 30 cycles of 30 sec at 94 ˚C/30 sec at 58 ˚C/2 min at 68 ˚C;
5 min at 68 ˚C final elongation time; PCR products were analyzed
on a 2% agarose TAE gel. The shorter fragment amplified only from
the proband was excised from the gel and sequenced on an ABI
3500HT (Applied Biosystems).

and Alamowitch, 2011] and CPK levels were persistently augmented [Tonduti et al., 2012]. Some peculiarities were found in
our patient: first of all, the neuroradiological abnormalities
showed an unusual evolution: they were not present at three
months of age, were evident at 30 months, and then improved on
subsequent imaging. In the majority of pediatric COL4A1
patients, lesions were documented or supposed to be related
to pre- or perinatal damage; in the pediatric setting, clinical
onset with stroke has, to date, been reported rarely and only in
adolescents [Shah et al., 2010]. No cases with clinical onset in
early infancy have been reported: the child here described is the
first case in which the vascular insult is hypothesized to have
occurred not in the pre-perinatal period but between three and
30 months of age. In fact, at three months of age his MRI was
completely normal, consistent with the physiological lack of
myelination at birth. We are not able to definitively confirm
the absence of cerebral calcification before three months because
unfortunately the child did not undergo neonatal cerebral ultrasound or CT. It is also underlined that at 3 months of age he
already showed microcephaly. On this basis we can only speculate that vascular events may have occurred between three and
30 months of age, in the absence of significant clinical symptoms,
a very unusual finding in infancy. The pattern of neuroradiological abnormalities found at the age of two and a half years was
characterized by small cystic changes in the left frontal white
matter, which could indicate a vascular insult, associated with
bilateral abnormal signal intensity, mainly in the periventricular
and deep white matter, compatible with diffuse small vessel
disease, and punctate calcification in the frontal periventricular
areas and the basal ganglia. Overall, these aspects are similar to
what has been previously reported in other COL4A1-mutated
subjects [Vahedi et al., 2003; Livingston et al., 2011; Vahedi and
Alamowitch 2011]. Moreover, during the neuroradiological
follow-up the extension and intensity of the lesions tended to
decrease progressively, particularly in the deep white matter.
This finding was not previously reported in COL4A1-mutated
patients, but in our opinion it could be compatible with the
occurrence of vascular insults, considering that the ischemic/
hemorrhagic damage due to vascular events in childhood can be
less pronounced than in adulthood and in pediatric patients may
improve over time [Kato et al., 2012].
In conclusion, this report of a novel pathogenetic mutation in the
COL4A1 gene provides evidence that brain lesions in COL4A1mutated patients can be asymptomatic, and can change and
improve over time. We also underline the importance of considering the possibility of COL4A1 mutations not only in the presence of
a pre-perinatal ischemic event, but also in the etiology of early

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childhood strokes. In this respect, the discovery of a cataract or of
raised CPK levels, as described in this patient, is a crucial element
evoking COL4A1 involvement.
Another remarkable aspect of this case is that the child can be
considered a compound heterozygote for two COL4A1 mutations:
indeed, our patient carried also a second variant in COL4A1 in
addition to the one already discussed. This second variant was
inherited from his father and was of unknown significance. A
polymorphism or a hypomorphic variant can be considered improbable because of the low frequency of the allele; this variant, too, was
predicted to be “damaging” by at least one of the in silico prediction
tools we used. We can therefore hypothesize additive effects of these
two mutations: the fact that the father suffers from Raynaud’s
phenomenon, muscle cramps and slightly raised CPK levels (despite
presenting a normal brain MRI and normal renal and ocular
phenotype) may suggest that this variant is related to his phenotype
and contributes to the unusual picture presented by his son.

ACKNOWLEDGMENTS
The Authors would like to thank Dr. O. Manara and Dr. C.
Agostinis from Bergamo and Dr. C. Parazzini and Dr. A. Righini
from Milan for providing first neuroradiological images of the
patient. This work has received funding from the Regione Lombardia government as an Independent Research Project (SVE-LA
Project, DGRS 13465–22/12/2010). We also acknowledge PRIN
2010–2011 (20108WT59Y_003) to O.Z.

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