Cognitive impairment, Epilepsy and
Brain Atrophy in two families with
different severities and outcomes
1
2
2
2
1
G. Cappuccio, G. Frisso, C. Cozzolino, F. Salvatore, E. Del Giudice
1
Department of Translational Medical Sciences, Section of Pediatrics
University of Naples Federico II
2
CEINGE-Biotecnologie Avanzate, Department of Biochemical and
Medical Biotecnologies
Family pedigree
I
II
III
3
4
5
6
ANTONIO TERESA
PIO
7
ROSA
Case Report
When 16 month-old seizures with
loss of consciousness, lateral
deviation of the head and eyes and
clonic jerks lasting ten minutes, one
episode per day
Two months later extensor type
infantile spasms
Delay in psychomotor milestones from
the beginning. Loss of achieved skills such
as independent ambulation. Presently she
can walk with support
Diffused brain atrophy
Rosa
Valproic acid,
seizure improvement
Hypsarrhythmia
ACTH
Lamotrigine
poor response
Topiramate
Good control
Case Report
Antonio Pio
Sleep EEG test
At 8 months infantile
spasms of the flexor type
ACTH followed by
Valproic Acid
Severe hypotonia and delay in
psychomotor milestones
Uncomplete control of the spasms
Recurrent episodes of apnea and comatose state
Brain Atrophy
Exitus
Case Report
Teresa
At eight months Flexor spasms
Valproic acid
Vigabatrin
Good seizure
control
At 16 months recurrence
of flexor spasms on awakening
Sleep EEG test
Topiramate
seizure free
Severe hypotonia and psychomotor retardation (complete head lag, unable to sit).
Global brain atrophy, dismyelinization
Neuroimaging
Rosa
Teresa
Different severity of fronto-temporal
atrophy and white matter abnormalities
Neuroimaging
Rosa
Teresa
Different degrees of brainstem and cerebellar
atrophy with corpus callosum hypoplasia
Neuroimaging
Rosa
Teresa
Vascular damages: ischemic lesions and dural ectasia with
bleeding
At this point
what did the biochemical
and genetic
exams reveal??
Investigations
Biochemical assays
Hcy
Aminoacidemia
and
Acylcarnitine
Ammonia, folate
and vitamin B12
Blood count Urine organic acid
and Urinary
oligosaccarides

Neg occasional
low Met
Normal
Neg
Neg

Antonio NA
Pio
Low Met
NA
Neg
Neg

Teresa
Neg occasional
low Met
Normal
Neg
Neg

Rosa

Urine
Hcy
Genetic tests
Karyoty
pe
Subtelomers
ArrayCGH
CDG
Angelman
methylation and
UBE3A test
ARX and
CDKL5
gene
Rosa
Neg
Neg
NA
Neg
Neg
NA
Antonio Pio
Neg
NA
NA
NA
NA
NA
Teresa
Neg
NA
Neg
Neg
NA
Neg
Shared phenotypical and
biochemical features
Rosa
Teresa
Hyperhomocysteinemia
•Epileptic
encephalopaty
•Microcephaly
•Cognitive
impairment
•Brain atrophy
Antonio
Pio
More severe
presentations
Hyperhomocysteinemia
Methymalonic aciduria
present
Megaloblastic anemia
present
Normal Cbl
Normal
TC
cblC
cblD
cblF
Low
TC
Low Cbl
IF
deficiency
Methymalonic aciduria
absent
Megaloblastic
anemia
absent
Normal/Low
Met
MTHFR
deficiency
High
Met
CBS
deficiency
TC
deficiency
Abnormal
processing
sample,
Respiratory
distress, MTHFR
polimorphisms,
Leukemia,
Hyothyroidism,
Epilepsy
Megaloblastic
anemia
present
Cbl
Low
Normal
IF
deficiency
TC
Normal
Low
TC deficiency
cblG
cblE
Differential Diagnosis
Enzyme/cofactor
Homocysteine
Methionine
Methylmalonic Macrocytosis
acid
Cystathionine βsynthetase
deficiency
Increased +++
Increased
Normal
Absent
MTHFR deficiency Increased +++
Low/LowNormal
Normal
Absent
Cobalamin C, D, F
Increased
Normal
Increased
Present
Cobalamin E, G
Increased
Normal
Normal
Present
Folate deficiency
Increased
Normal
Normal
Present
Nutritional
cobalamine
deficiency
Increased
Normal
Increased
Present
Remethylation defects
Bishop et al., 2008; Schiff et al. 2011
Remethylation Defects
MTHFR: Methylenetetrahydrofolate reductase
CblE: Methionine synthase reductase
CblG: Methionine synthase
CBS: Cystathionine-β-synthase
Remethylation Defects
Clinical features
Early onset
Onset range between 1-10 years
Hypotonia, Letargy
Cognitive impairment, Ataxia
Microcephaly
Seizures
Seizures
Fatigue
Coma, Apnea
Spasticity, Piramidal signs
Hematological anomalies
Thrombotic events
Ectopia lentis
Adult onset
Similar findings
Peripheral signs
Psychiatric signs
Molecular analysis of MTHFR gene
MTHFR gene
• At 1p36.3
• Lenght 20 kb
• 12 exons
• mRNA 7150 bp
Exons/intron and of 5’- and 3’UTR of MTHFR gene
DNA extraction from blood
samples (Salting out)
Molecular analysis of MTHFR gene
Direct Sequencing of PCR Products
Results for Teresa & parents
Emilia
Luigi
c.547C>T
(p.R183X)/ WT
WT/ c.1013T>C
(p.M338T)
Teresa
c.547C>T (p.R183X) / c.1013T>C (p.M338T)
Molecular analysis of MTHFR gene
Results for Rosa & parents
Luigi
Emilia
p.R183X/
WT/
WT
p.M338T
Teresa
p.R183X/
p.M338T
Luisa
Massimiliano
WT/
c.547C>T (p.R183X)/
c.1013T>C (p.M338T)
WT
Rosa
c.1013T>C (p.M338T) / c.547C>T (p.R183X)
Molecular analysis of MTHFR gene
Luigi
Emilia
p.R183X/ WT
WT/ p.M338T
p.A222V/WT
p.A222V/WT
p.F435F/p.F435F p.F435F/p.F435F
Teresa
p.R183X/ .M338T
p.A222V/WT
p.F435F/p.F435F
Luisa
WT/ p.M338T
p.A222V/WT
p.F435F/p.F435F
Massimiliano
p.R183X/ WT
p.F435F/p.F435F
Rosa
p.M338T / p.R183X
p.A222V/WT
p.F435F/p.F435F
Severe MTHFR Deficiency
• MTHFR is the most prevalent inborn error of folate metabolism.
More than 100 cases of MTHFR deficiency have been reported.
• MTHFR deficiency results from the disruption of enzyme function
less than 20% of controls.
• Wide range of clinical manifestations from asymptomatic to severe
psychomotor retardation, epilepsy, microcephaly. Sometimes
central respiratory failure can be responsible for death. In the late
childhood or adulthood arterial thrombosis or psychiatric
disturbances can be the initial signs.
• Diagnosis based on evaluation of serum Hcy and low normal
level of Methionine in the absence of hemetological signs and
Methylmalonic aciduria
Therapy
Betaine (Cystadane)= 100-250 mg/Kg-6-9 gr/die
Folinic acid (Citofolin)= 1.55 mg/kg/die
B12 Vitamin (Dobetin)= 50-150 µg/die
B6??
Therapy
Rosa
Therapy
Al Tawari et al., 2002, Schiff et al., 2011, Saudubray et al. 2006
Teresa
Identical genotype vs variable
phenotypic expression
•
Previous reports described intrafamilial phenotypic variation Tonetti et al., 2001; Tallur et al., 2005;
•
-
Outcome enviromental modifiers
Treatment timing
Antiepileptic drugs (Phenytoine, Carbamazepine)
Arai and Osaka, 2011, Al Essa et al., 1999,
Forges e t al., 2010
- Nitrous oxide
Selzer at al., 2003;
- Combined defects in more
than one enzyme involved
in remethylation pathway
• Different ability in Detoxification of Hcy-metabolites such as Hcy-thyolactone
- Bleomycin hydrolase and Paraxonase 1 significantly contribute to Hcy-thyolactone
metabolism protecting against neurodegeneration Borowczyk et al., 2012;
•
Age related onset of epileptic encephalopathy
NEUROTOXICITY IS A MULTIFACTORIAL EVENT!
Epileptic
Encephalopathy
Prenatal
disorders
Neurocutaneous disorders
Tuberous sclerosis, Sturge-Weber disease,
Incontinentia pigmenti, Neurofibromatosis
Chromosomal abnormalities
Down syndrome, Miller-Dieker syndrome
Aicardi syndrome, Agyria (lissencephaly),
pachygyria, polymicrogyria, Schizencephaly
Hypoxic-ischaemic encephalopathies, Congenital infections
Hypoxic-ischemic encephalopathies, Infections, Trauma and Intracranial haemorrhage
Malformations of cerebral development
Perinatal
disorders
Pyridoxine dependency, Non-ketotic hyperglycinaemia,
Phenylketonuria, Maple syrup urine disease, Mitochondrial
encephalopathies
And MTHFR deficiency!!!!
Infections (meningitis, encephalitis)
Metabolic
Postnatal
disorders
Degenerative diseases, Drugs
Epileptic Encephalopathies in Infancy and Early Childhood, 2007
Take-home messages
• MTHFR deficiency is a rare entity. Infantile spasms are a possible initial
manifestation.
• Its important not to miss such disorders because specific and early treatment
interventions are possible. The neurological outcome is more favorable
earlier the treatment is administered.
• Diagnostic work-up is simple and inexpensive.
• Neurotoxicity in MTHFR deficiency is a multifaceted process in which the
hyperhomocysteinemia may be considered a trigger and not a primary
cause, increasing the susceptibility of CNS to other indipendent influences.
Merci à tout le monde
The aim of medicine is to
know the disease….
..to relieve the sufferings
it causes
Main clinical problems
Rosa
12 years and 5
months
• Epileptic encephalopathy
• Microcephaly
• Severe cognitive impairment
• Brain atrophy
Geni modificatori???
Identificati SNPs in geni implicati nel metabolismo dell’acido
folico e della vitamina B12. Di questi, il gene ALDH1L1 (membro
della famiglia aldeide deidrogenasi), si è dimostrato interagire con
MTHFR
Main clinical problems
Antonio Pio
Exitus at
16 months
Teresa
3 years and 7
months
•
Epileptic encephalopathy
•
Epileptic encephalopathy
•
Mycrocephaly
•
Mycrocephaly
•
Hypotonia
•
Severe cognitive impairment
•
Brain atrophy
•
Congenital hypothyroidism
•
Acute respiratory distress
•
Brain atrophy
MTHFR deficiency
• Più frequente disordine del metabolismo dei folati, AR
• I polimorfismi in cis rispetto alla presenza di
mutazioni patogeniche possono portare ad una
riduzione dell’attività enzimatica
• Neurotossicità cerebrale associata ad un aumento di
Hom e riduzione die livelli di SAM Severità del
fenotipo correla con la riduzione dell’attività
enzimatica
• I segni iniziali sono per lo più neurologici
Mutazione p.R183X in MTHFR
• Mutazione nonsenso
• Grave fenotipo clinico
• Assenza di attività enzimatica della proteina
MTHFR
Mutazione p.M338T in MTHFR
• Mutazione missenso che causa alterazione del
folding della proteina
• Ridotta attività enzimatica della proteina
MTHFR
I polimorfismi di MTHFR
MTHFR p.F435F
MTHFR p.A222V
Famiglia MTHFR
I. Luigi
p.R183X/ WT
C. Luisa
C. Massimiliano
C. Giuseppe C. Stefania
WT/ p.M338T p.R183X/ WT WT/ p.M338TWT/ p.M338T
C. Emilia
WT/ p.M338T
C. Rosa
p.R183X/ p.M338T
I. Assunta
I. Vincenzo
WT/p.M338T WT/p.M338T
I. Teresa
p.R183X/ p.M338T