Accepted Manuscript

Clinical and genetic studies in patients with Lafora disease from

Pakistan

Arsalan Ahmad, Rubina Dad, Muhammad Ikram Ullah, Tahir

Ahmed Baig, Imran N. Ahmad, Abdul Nasir, Christian A. Hbner,
Muhammad Jawad Hassan

PII: S0022-510X(17)30008-4
DOI: doi: 10.1016/j.jns.2017.01.010
Reference: JNS 15072
To appear in: Journal of the Neurological Sciences
Received date: 2 June 2016
Revised date: 29 December 2016
Accepted date: 3 January 2017

Please cite this article as: Arsalan Ahmad, Rubina Dad, Muhammad Ikram Ullah, Tahir
Ahmed Baig, Imran N. Ahmad, Abdul Nasir, Christian A. Hbner, Muhammad Jawad
Hassan , Clinical and genetic studies in patients with Lafora disease from Pakistan. The
address for the corresponding author was captured as affiliation for all authors. Please
check if appropriate. Jns(2017), doi: 10.1016/j.jns.2017.01.010

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 ACCEPTED MANUSCRIPT

Clinical and genetic studies in patients with Lafora disease from Pakistan

Arsalan Ahmada*, Rubina Dadb*, Muhammad Ikram Ullahc, Tahir Ahmed Baigb, Imran N.
Ahmadd, Abdul Nasire, Christian A. Hbnerf , Muhammad Jawad Hassanb**

a
Division of Neurology, Shifa International Hospital, Shifa Tameer e Millat University,
Islamabad, Pakistan
b
Department of Healthcare Biotechnology, Atta-ur-Rahman School of Applied Biosciences

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(ASAB), National University of Sciences & Technology (NUST), Islamabad, Pakistan

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c
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University,
Islamabad, Pakistan

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d
Division of Pathology, Shifa International Hospital, Shifa Tameer e Millat University,
Islamabad, Pakistan
e
Department of Biochemistry, Abdul Wali Khan University Mardan, Pakistan

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f
Institute of Human Genetics, University of Jena, Kollegiengasse 10, D-07743 Jena, Germany
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*These authors contributed equally to this work, so both will be considered as first author.
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**Corresponding Author:
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Muhammad Jawad Hassan PhD

Assistant Professor Healthcare Biotechnology
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Atta-ur-Rahman School of Applied Biosciences (ASAB)

National University of Sciences & Technology (NUST)
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Sector H-12, Islamabad, 44000, Pakistan

Phone: +92 51 9085-6135, Email: mjh@asab.nust.edu.pk
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ABSTRACT

Lafora disease (LD) is progressive myoclonic epilepsy with late childhood- to teenage- onset.
Mutations in two genes, EPM2A and NHLRC1, are responsible for this autosomal recessive
disease in many patients Worldwide. In present study, we reported two unrelated consanguineous
Pakistani families with Lafora disease (Families A and B). Affected individuals in both families
presented with generalized tonic clonic seizures, intellectual disability, ataxia and cognitive

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decline. Diagnosis of Lafora disease was made on histo-pathological analysis of the skin biopsy,

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found positive for lafora bodies in periodic acid schiff stain and frequent generalized
epileptiform discharges on electroencephalogram (EEG). Bi-directional sequencing in Family A

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was performed for EPM2A and NHLRC1 genes but no mutation was found. In family B, Ilumina
Trusight one sequencing panel covering 4813 OMIM genes was carried out and we identified a

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novel homozygous mutation c.95G>T; p.32Trp>Leu of EPM2A gene which was found co-
segregated in this family through sanger sequencing. Structural analysis of this mutation, through
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different in silico approaches, predicted loss of stability and conformation in Laforin protein.

Key words: Lafora disease, Mutation, EPM2A, Pakistani families

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1. Introduction
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Lafora disease (LD) is progressive myoclonus epilepsy (PME) of adult onset inherited in
autosomal recessive manner (MIM-254780). Cellular presentation includes characteristics
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intracellular inclusion bodies (Lafore polyglucosan bodies) after pathognomonic periodic acid-
schiff (PAS) staining [1]. Besides axons and dendrites of the central nervous system, these large
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basophilic PAS inclusions, are also found in the retina, heart, liver, muscle, and skin [2]. LD is
classically described as symptoms starting in early adolescence as stimulus sensitive generalized
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tonic-clonic, absence, visual, and myoclonic seizures [3]. The cardinal features of LD include
progressive dementia, psychosis, cerebellar ataxia, dysarthria, amaurosis, mutism, muscle
wasting, and respiratory failure lead to death within 10 years of the onset [4, 5]. LD, although
relatively rare in the developed countries, is commonly encountered in the Mediterranean and
Asian countries where a high rate of consanguinity is practiced [2]. Management and cure is still
a challenge for this disorder.
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Till present, about three loci are implicated in LD so far. The first one, EPM2A, was
identified in late nineties [6, 7] and the second gene, NHLRC1 was discovered in the year 2003
[8]. EPM2A is located on chromosome 6q24 and encodes laforin, a dual-specificity protein
phosphatase having 331 amino acids and a functional carbohydrate-binding domain at the N-
terminus. Second gene NHLRC1, also known as EPM2B, is located on chromosome 6p22.3 and
encodes malin, an E3 ubiquitin ligase of 395 amino acids with a RING finger domain at the N-
terminus and six NHL domains in the C-terminal region [9, 10] A third gene, PRDM8, has also

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been reported that codes for a protein possibly interacting with laforin and malin and that could

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be expressed in a childhood-onset sub-form of Lafora disease [11].

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Defects in EPM2A and NHLRC1 accounted about 80% of the LD families that are screened
for genetic lesions. In the remaining families, a role for a new gene is suggested [9, 12-13].

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Various reports about mutations in EPM2A suggested that the course of the disease was longer in
patients with EPM2B mutations compared to patients with EPM2A mutations, implying that
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patients with EPM2B-associated LD tend to have a slightly milder course and slower progression
[1, 5, 14-16]. The onset and progression of the disease may vary due to genetic heterogeneity and
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diversity of mutations, making genotype-phenotype correlations difficult [1, 5]. Ganesh et al.
(2002) studied 22 patients with LD and described two clinical types of the disease: (1) classic LD
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started in adolescence with stimuli sensitive myoclonic, tonic-clonic, absence and occipital
seizures followed by dementia and rapid progressive neurological deterioration [17]. Classic LD
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was mainly associated with mutations in exon 4 of EPM2A; and (2) atypical LD starts in
childhood with dyslexia and learning disorders followed by epilepsy and rapidly developing
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neurological impairment. Atypical LD was found associated with mutations in exon 1 of EPM2A
gene [18].
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In this report, we presented three affected individuals from two consanguineous families
showing symptoms of Lafora disease for clinical and genetic identification of mutation by
Sanger sequencing and Target sequencing panel. In family A, no mutation was found in adult
onset LD genes EPM2A and NHLRC1(EPM2B, while a novel homozygous mutation c.95G>T;
p.32Trp>Leu (W32L) was detected in family B. Protein structural analysis identified loss of
stability and conformation in laforin protein due to this mutation.
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2. Patients and methods

After ethical approval, two families who visited to Neurology Division of Shifa International
Hospital, Islamabad, were recruited for this study. Family A consisted of one affected individual,
belonged to Punjabi language group and family B comprised of two affected individuals
belonged to Siraiki language group. Blood was collected after informed consent.

2.1 Mutation detection by TruSight One Sequencing Panel and Sanger Sequencing

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Genomic DNA was extracted from peripheral venous blood using the QIAmp DNA
blood Mini Kit (QIAGEN, Valencia, CA, USA). In family A, coding regions and exon-intron

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boundaries of EPM2A and NHLRC1 genes were Sanger sequenced by using DTCS Quick Start
sequencing kit on CEQ8000 Genetic analyzer (Beckman Coulter, Fullerton, CA, USA) according

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to standard protocols. (Primers sequences are available on request).

Targeted sequencing was carried out in one affected individual of Family B by using
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trusight sequencing panel technique for genes of neurological disorders (especially of epilepsy
phenotype). We performed enzyme based library preparation of genomic DNA. Target
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enrichment was done using the Illumina TruSight One Sequencing Panel followed by NGS using
the HiSeq 2500 [Illumina, Inc. San Diego, CA]. This kit enriches for 62,000 exons from 4,813
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genes, producing a collective target region size of 12 Mb. Clinical testing was ordered as pre-
defined gene panels. DNA was extracted from peripheral blood (Qiagen, Valencia, CA) and 50
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ng was used for Nextera-based library preparation following manufacturers protocols

(Illumina). Four clinical samples and one HapMap control were pooled and run across two lanes
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on a HiSeq 2500 for an average of 5 samples per lane. For targeted clinical panels (<50 genes),
areas with <15X coverage were routinely Sanger sequenced to achieve adequate analytic
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sensitivity [19]. Sanger sequencing was performed for exon 1 of EPM2A gene to confirm the
mutation in family B detected through panel sequencing.

2.2 Comparative Protein modeling

The effect of the mutation (Trp32Leu) on the structure and function of the Human
Laforin protein was analyzed. The x-ray structure of Human Laforin (PDB id: 4RKK) was
retrieved from Protein Data Bank [20]. The structure for Trp32Leu mutant was developed by
changing the selected residue into desired residue by using similar refinement of the structure
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and energy minimization protocol that was used for the wild-type structure. The potential energy
of the wild-type and mutant proteins was compared. The refined structures were used to study
the effect of mutation on the Laforin structure. The protein structure was inspected using
PYMOL viewer (http://www.pymol.org).
3. Results

3.1Clinical Description

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3.1.1 Patient (IV:2) of family A:

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This 25 year old female, born to consanguineous parents (Fig. 1A), first presented to

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neurology outpatient department (OPD) in March 2004 with a two year history of episodes of
flashes of light like shining stars followed by headache and nausea which lasted for 2-3 hours as

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well as a single generalized tonic clonic seizure. She was a student of grade 8 and of tall stature
and medium built. EEG done previously showed focal spike wave activity. She was taking
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Sodium Valproate (VPA) 125 mg BD (twice a day). On examination, her higher mental function
speech and gait were normal. There was mild temporal pallor bilaterally on fundoscopy. Extra
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ocular movements were full. There is no focal limb weakness. Deep tendon reflexes were brisk
bilaterally. Planter responses were bilateral flexor. Differential diagnosis at that time included
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Migraine with aura vs occipital lobe epilepsy. She was advised MRI of the brain without contrast
which was normal. Tablet Topiramate was added in 2004 when she had a second generalized
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seizure. When seen in again June 2004 she complained of single jerks/myoclonic jerks as well as
single atonic seizure that improved with clonazepam. Her seizures continued and she gained
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weight with VPA. Lamotrigine (LTG) was added in August 2004. By 2009 she still had a few
myoclonic jerks/week and 1-2 generalized tonic clonic seizures/month. She was wheel chair
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bound. Medication at that time were LTG 100 mg BD, Primidone 250 mg at night, Epival (VPA)
375 mg BD, Levitracetum (Lev) 375 mg BD, Clonazepam (Czm) 2 mg TID (three times a day)
and Phenytoin (Pht) 150 mg BD.

In December 2012 she was bought to the emergency room in status epilepticus. She
needed to be intubated and placed on ventilator. EEG showed frequent generalized epileptiform
discharges. Despite, multiple medications seizures could not be controlled so capsule
Zonisamide (Zsm) 50 mg BD increasing to 100 mg BD and tablet Piracetam 800 mg TID were
added. She was slowly weaned off the ventilator. Axillary skin biopsy for Lafora bodies showed
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presence of Lafora bodies (Figure 2. She was diagnosed as case of Lafora body disease (Fig.
1B). Her Genetic test for Lafora body disease was recommended. At present, she is on multiple
anticonvulsants. She still has 1-2 generalized tonic clonic seizures/month, in addition to multiple
myoclonic seizure early morning. She is mostly bed bound but is able to take oral feed. She also
has limited communication with family members.

3.1.2 Patient 1 (IV:1) of family B

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This 15 year old female was first seen in neurology OPD clinic in July 2014 with a one year

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history of seizures, slurring of speech for last 3-4 months, inability to walk and cognitive decline

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for last 2 months and frequent single jerks. One year back she had generalized tonic clonic
seizures. Later she started having frequent myoclonic jerks. Three to four months back she

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started to develop speech difficulty and slurring. She also had progressive cognitive decline. At
the time of presentation she was only able to speak short sentences. Her parents are maternal first
cousins, one younger brother also has similar disease while other sibs are normal (Fig. 1C).
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On examination, she was conscious, she had slurred speech and did not follow complex
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commands. She had action myoclonus, mild spastic weakness all four limbs, deep tendon
reflexes were ++ and symmetrical, plantar responses were bilateral flexor. Previously she had an
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MRI brain at an outside facility which was reportedly normal. Previous EEG showed generalized
poly spike and wave discharges with a moderate background slowing. Workup for Wilson
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disease was negative. Thyroid functions were normal. At that time her medication included tablet
VPA 1000 mg BD, LTG 100 mg BD, CZM 2 mg HS and LEV 250 mg BD.
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She appeared to be suffering from progressive myoclonic epilepsy, most likely Lafora body
disease. Her axillary skin biopsy showed intra-cytoplasmic inclusions consistent with Lafora
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bodies (Fig. 1D). MRI of the brain was repeated again and it showed mild cortical and
subcortical atrophy (Fig. 1E). LEV was increased to 500 mg in AM and 250 mg in PM. Tablet
Piracetam 800 mg TID was added. Prognosis was explained to the family. She presented again in
August 2014 increase frequency of myoclonic seizures. ZSM 50 mg OD (once a day) was added
and was slowly increased to 100 mg TID over the next month. She was also advised a
Gastrostomy tube. She still has 3-4 generalized tonic clonic seizures/month. Present medication
includes ZSM 400 mg/day, LEV 2,250 mg/day, LTG 200 mg/day, VPA 2000 mg/day, CZM 3
mg /day
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3.1.3 Patient 2 (IV:2) of family B:

This 12 years old boy presented to neurology OPD in November 2014 with a one year history
of seizures. First event was occurred after exertion/playing. VPA 250 mg BD was started. EEG
showed generalized spike and wave discharges. His elder sister is suffering from biopsy proven
Lafora Body disease. On examination, he was alert and oriented, speech was normal. Gait was
normal, cranial nerves examination was unremarkable. He had no limb weakness, deep tendon

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reflexes were ++ and equal, plantar reponses were bilateral flexor. Valporate was increased to

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375 mg BD. He was seen again in May 2015 when he complained of 2-3 myoclonic seizures.
EEG showed frequent generalized poly spike and wave discharges as well as some symmetrical

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6 Hz theta activity seen over bilateral occipital regions. VPA was increased to 500 mg BD, one
tablet POD. In 2016, Zonisamide 100 mg BD was added. He is presently independent in

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activities of daily livings but has left school.

3.2 Genetic analysis and Mutation detection

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In family A, DNA sequence analysis of the EPM2A and NHLRC1 gene did not reveal any
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pathogenic variant in coding regions. In family B, through target sequencing a novel mutation
(c.95G>T; p.32Trp>Leu) of EPM2A gene was identified and co-segregation by Sanger
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sequencing confirmed the mutation in this family. The homozygous peak with mutation c.95G>T
was present in both patients (IV:1 and IV:2), the carrier parent (III:3) showed heterozygous peak
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and unaffected family member (IV:3) was a wild-type. Sequencing images of one affected, one
carrier and a normal individual have been shown (Fig. 2A-2C). The variant was absent in 100
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control chromosomes of the Pakistani population.

3.3 Protein Homology analysis
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In the native structure, the Tryptophan residue, located in the loop region is involved in
the intermolecular interaction. Tryptophan possesses an indole group and change with leucine
having short aliphatic group lack intermolecular bonding capabilities unlike tryptophan. This
substitution resulted in the loss of intermolecular bonding. Hence, the mutant was less stable
which was supported by increased in potential energy (-2530.352 kcal/mol) as compared to wild-
type Laforin (-2602.488 kcal/mol). The Trp32 in wild-type Laforin forms two intra-molecular
bonds with the side chain of nearby Trp99 and Trp85 residues (Fig. 2D-2H).
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4. Discussion

Lafora disease is one of the rare neuro-developmental diseases of early adolescent. It is

presented with diverse clinical features like abnormal social interactions, continuous seizures,
ataxia and intellectual disability, decline of neuronal functions and loss of motor movements at
later stages of the disease [21]. Worldwide, the approximate prevalence of epilepsy is 8% in
general population while in developing countries a higher burden of this disease is found with

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rate of prevalence between 10 and 40% [22, 23]. Similarly, in Pakistan prevalence is presumed to

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be high but no data is available.

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In present study, we reported two consanguineous Pakistani families of Lafora disease
evident with positive lafora bodies in skin biopsy. Inside the skin, Lafora bodies have been

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obtained in biopsies from the axilla or from skin outside the axilla. Interestingly, the cell type
found to be affected in each of these two locations is different, ducts of ecrine glands (former)
and myoepithelial cells surrounding apocrine ((later). Andrade et al (2003) are of the view that a
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biopsy of skin outside the axilla for the diagnosis of LD should be preferred as in the absence of
DNA testing facility in most of the clinics; histopathology would be the method of choice in LD
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diagnosis [24].
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In both families, parents were first cousins (Figure 1). In all affected kindred, EEG showed
frequent generalized epileptiform discharges. Axillary skin biopsy for lafora bodies was
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performed with PAS stain and it was positive for lafora bodies in affected individuals both
families (Figure 1). Genetic analysis of these families by identified a novel mutation (c.95G>T;
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p.32Trp>Leu) of EPM2A gene only in family B while no mutation was found in family A for
previously known genes (EPM2A and NHLRC1) of Lafora disease. Previously, only one case
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report of one Pakistani patient with LD is available [25]. We present detailed reports of clinical
investigations and molecular analysis in two families.

Different types of mutations are associated with lafora disease including micro-deletions,
insertions, missense and frame-shift mutations resulting in malfunctioned Laforin or Malin
leading to development of LD. Most mutations in lafora disease are missense mutations either
affecting carbohydrate binding domain or dual phosphatase domain of laforin or affecting RING
domain or NHL repeats of malin. At present, about 73 mutations are reported in EPM2A gene
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and 40 of them are missense mutations; about 58% of total mutations in these two genes (HGMD
Professional 2015.2),

EPM2A mutations are diverse and heterogeneous distributed in different populations of the
world including Caucasian (European origin), Asian (Indian, Pakistan, Japanese, and Korean)
and Arabic (countries of the Middle East) populations. All missense mutations are anticipated to
be loss of function mutations, as the resulting phenotype is similar to the patient group that had

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larger deletions [17]. The larger deletions of the EPM2A gene are quite frequent in the Arabic

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population as compared to other types of mutations. The frequencies of four distinct types of
mutations in the other two populations were more or less comparable. Unusually, larger deletions

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in the Arabic population are limited to the first exon of the EPM2A gene [1] while deletions in
exon 3, are unique to Indian population [12].

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In family A, no mutation was identified in coding regions and exon-intron boundaries of
EPM2A and NHLRC1 genes. We are short of blood/DNA for this affected individual and due to
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continuous ill condition of only affected individual, parents/guardians were not allowing re-
sampling. Therefore, sequencing of PRDM8 gene, containing a mutation previously identified
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from Pakistan is not possible at the moment.

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Comparative protein analysis of c.95G>T, W32L mutation predicted that Tryptophan residue
is involved in the intermolecular interaction. Tryptophan possesses an indole group and its
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replacement with leucine resulted in loss of intermolecular bonding due to its short aliphatic
group that lacks intermolecular bonding capabilities unlike tryptophan. The mutant protein is less
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stable due to increase in potential energy (-2530.352 kcal/mol) as compared to wild-type Laforin
(-2602.488 kcal/mol). The Trp32 in wild-type Laforin forms two intra-molecular bonds with the
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side chain of nearby Trp99 and Trp85 residues (Figure 2). Due to loss of intra-molecular
interactions and structural bonding, perturbation of the loop conformation in the protein is
observed. Earlier, mutation of same codon W32G; was tested for molecular dynamics (MD) to
check the conformational function of Laforin structure due to this mutation. Different parameters
of MD observed structural alterations in atomic level of mutation W32G. Consequently, these
conformational changes led to the loss of protein stability [26].

In conclusion, we report three patients in two consanguineous Pakistan families with clinical,
histological and genetic analysis of LD. In one family, a novel mutation c.95G>T (p.Trp32Leu)
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was identified and protein structural analysis depicted increased in potential energy and loss of
intra-molecular bonding. In second family no pathogenic variant was identified in EPM2A and
NHLRC1 genes. For this family, a previously known mutation of PRDM8 gene must be checked
before a whole exome sequencing/target panel sequencing [11]. This report may establish that
there is huge heterogeneity in rare disorders like Lafora disease in Pakistani population.

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Acknowledgments: We are grateful to the families who participated in this research. Rubina

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Dad was supported by HEC-IRSIP program from Pakistani government for her PhD research.

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Conflict of Interest: All authors declare no conflict of interest.

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Figure Legends
Fig. 1. Pedigree of a family A with member suffering from Lafora disease (A) Axillary skin
biopsy of affected female of family A showing apocrine sweat gland with cytoplasmic Lafora
body (arrow) (PAS stain, 40x) (B) The pedigree of family B is outlined showing affected and
unaffected individuals (C). Axillary skin biopsy in affected female of family B showing apocrine
sweat gland with cytoplasmic Lafora body (arrow) (PAS stain with silver, 40x) (D) MRI brain of
affected female of family B showing cortical and sub-cortical atrophic changes (E)

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Fig. 2. DNA sequence of EPM2A represents the affected individual (IV:1) of family B with
homozygous peaks of novel c.95 G>T (A), carrier father (III:3) is heterozygous (B) and

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unaffected brother (IV:3) with normal sequence (C). Representation of predicted structure for
human Laforin, region of mutation is highlighted by circle (D) Predicted secondary structure

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pattern of wild-type Laforin (E) and predicted computed surface of wild-type Laforin (F),
Predicted secondary structure pattern of mutant Laforin (G) and predicted computed surface of
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mutant Laforin (H).
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Highlights

Clinical and genetic studies in patients with Lafora disease from Pakistan

We recruited three patients from two consanguineous families for clinical and genetic analysis of
autosomal recessive Lafora disease from Pakistan. Affected individuals showed typical features of
Lafora disease with PAS positive lafora bodies on histological biopsy. Bi-directional and the
Ilumina True sight One panel sequencing identified a mutation in EPM2A gene in family B and

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precisely segregates with the disease in this family, as confirmed by Sanger sequencing. On the

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other hand, direct sequencing of EPM2A and NHLRC1 genes in family A did not reveal any

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nucleotide variation. Protein modeling studies of mutation in family B suggested a disruption in
spatial configuration of mutant Laforin. In accordance of published data, this is the first report of

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EPM2A gene mutation in Lafora disease from Pakistan.
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