In the cohort under study, a total of eight patients were enrolled, with ages spanning from 4-6 years. The studies group participants consisted of 5 females and 3 males. All the patients included in this study were from distinct families and were unrelated to each other. The collected dataset is described below and encompasses the demographic information, genotypic profile, key clinical, neuroimaging and electrophysiologic characteristics. Among the clinical phenotypic spectrum, our group focused on studying the onset and progression of neurologic symptomatology and comprehensive neuropsychological evaluation.
Demographics/Genotype:
All 8 patients were clinically diagnosed with CLN7 deficiency, and the diagnosis was molecularly confirmed with homozygous or bi-allelic heterozygous pathogenic mutations in MFSD8 gene. The genetic diagnosis was made through epilepsy gene panels, whole exome sequencing and MFSD8 sequence analysis. Descriptive analysis was used to analyze the data which revealed heterogeneity of geographic, cultural, and linguistic and demographics backgrounds as detailed in Table 2.
Table 2: Demographics- participants gender, country of origin and genotype
Subject ID
|
Gender
|
Country/
Language
|
Affected Gene
|
Mutation Information
|
Zygosity
|
Predicted DNA Variation
|
1
|
Female
|
India-USA/
English
|
MFSD8
|
c.1351-2A>G
|
Homozygous
|
Splice acceptor
|
2
|
Male
|
USA/
English
|
MFSD8
|
c.1036delG (p. Val346LeufsTer68)
c.440-2A>T
|
Compound Heterozygous
|
Frameshift
Splice acceptor
|
3
|
Female
|
Sweden/
Swedish
|
MFSD8
|
c.1437G>A,
(p.Trp479Ter)
c.1444C>T
(p.Arg482Ter)
|
Compound Heterozygous
|
Stop gain
Stop gain
|
4
|
Male
|
Hungary/Hungarian
|
MFSD8
|
c.881C>A,
p.(Thr294Lys)
c.754+2T>A
|
Compound Heterozygous
|
Missense
Splice donor
|
5
|
Female
|
Paraguay/
Spanish
|
MFSD8
|
c.103C>T, (p.Arg35Ter)
|
Homozygous
|
Stop gain
|
6
|
Male
|
Romania/
Romanian
|
MFSD8
|
c.1373C>A, (p.Thr458Lys)
|
Homozygous
|
Missense
|
7
|
Female
|
Saudi Arabia/
Arabic
|
MFSD8
|
c.198+2T>C
|
Homozygous
|
GT Splice donor
|
8
|
Female
|
USA/
English
|
MFSD8
|
C.1444C>T
(p. Arg482Ter)
C.1206del (p.Ile403LeufsTer11)
|
Compound Heterozygous
|
Stop gain
Frameshift
|
Clinical Characteristics:
Birth history, early development, and age of onset of symptoms:
All patients (8 out of 8) were reported to have an uncomplicated birth and an unremarkable early post-natal period. Early development was normal until 2 years of age.
The earliest symptom onset was at two years of age. In all the patients (8 out of 8) the onset of symptoms was noted before 4 years of age (Table 3) and the age of diagnosis was between 4 and 5 years. In our study cohort, median age of onset of symptoms is 3.4 years and median age of diagnosis is 4.8 years.
In the study cohort, the initial presenting symptoms prompting the families to seek medical attention varied among the patients. Professedly, 2 out of 8 patients presented with gait disturbance as the initial symptom, while another 2 out of 8 exhibited signs of tremors and recurrent falls. Additionally, one patient presented with vision changes, and difficulties in language were reported in 2 out of 8 patients, either in terms of articulation or fluency and 1 out of 8 patients was brought to medical attention because family was concerned about developmental delays, which was described as an overall concern about the lack of progress in neurologic development specifically in language and fine motor domains.
It is noteworthy, that the first clinical symptoms, prompting the family to seek medical attention is parent reported and is described here as it was noted in medical records. It is important to mention that detailed clinical and neuropsychological assessments were not conducted at the time of onset to provide detailed characterization of specific nature and extent of language difficulties or developmental delays.
Another noteworthy observation emerged during retrospective analysis, that the majority of patients, 6 out of 8 to be specific, had a paucity in language development, occurring at or right after 2 years of age. This plateau in language development, however, did not attain a level of significance which would either prompt families to seek medical attention or alert the primary care physician to pursue further diagnostic testing.
Neurological Symptoms
a. Developmental Regression:
Gait Problems:
Gait disturbance was reported by families as early as 2 and a half years of age and as late as 4 and half years of age. Gait progressively deteriorated requiring an aid to walk within 1-2 years from onset of gait difficulty (Table 3). Once walking difficulties were noticed, the earlier level of function was never obtained. All the patients who were seen at advanced disease stage were non-ambulatory within 1 year of age when they needed help with walking (Figures 1 and 2).
Language Difficulties:
Language difficulties or delays were noted as early as 2 years of age in 3/8 patients, 2.5 years in 3/8 patients, 3.5 years in 1/8 patients and by 4 years in 1/8 patients. All the patients at the time of visit had moderate to severe language issues.
b. Seizures:
In this patient cohort the onset of seizures ranged from 3 years and 5 months to 4 years and 10 months. Various seizure types were reported including myoclonic, atonic, and bilateral tonic-clonic seizures. In addition to seizures, myoclonic jerks were reported in all but one study participant.
In this study cohort, the majority (6 out of 8) of patients required more than one anti-seizure medication to achieve adequate seizure control. The most common anti-seizure medication used in this study cohort was Valproic acid, and 6 out of 8 patients were taking Valproic acid. In one patient, Valproic acid was stopped later in the disease course and replaced with an alternative medicine. Clobazam and Levetiracetam were the second most common medications used and 5 out of 8 patients were taking these anti-seizure medications. Lamotrigine was also used for seizure control in 2 out of 8 patients in this cohort. Cannabidiol was used in 2 out of 8 patients. One study patient was on Rufinamide and Clonazepam. There was no clear benefit of one anti-seizure medication over the other, however, this data is not sufficient to establish such a correlation. One patient had excessive sedation on combination of Clobazam and Cannabidiol, however there were no other harmful effects or side effects reported for any other anti-seizure medication at the time of the study. It should be noted that one of the limitations of the retrospective study is paucity of data and a complete profile of longitudinal use of anti-seizure medications along with the side effects and interactions of various medications is not available to report in some cases.
Table 3: Developmental regression
Subject ID
|
Age at Visit (yy.mm)
|
Age at Onset of first symptom (yy.mm)
|
Age at Diagnosis (yy.mm)
|
Initial Symptom
|
Seizure onset (yy.mm)
|
Onset of Gait Problems (yy.mm)
|
Onset of Language difficulties (yy.mm)
|
Onset of Cognitive issues (yy.mm)
|
Onset of Vision Changes (yy.mm)
|
1
|
6.9
|
3.4
|
4.4
|
Gait Problems
|
4
|
3.5
|
4
|
3.5
|
5
|
2
|
6.11
|
4
|
4.75
|
Vision change
|
4.4
|
4.5
|
2.5
|
Unsure
|
4
|
3
|
5.5
|
2.4
|
5
|
Gait problems
|
4.8
|
2.5
|
2.5
|
3.5
|
2.5
|
4
|
5.6
|
3
|
5
|
DD
|
3.5
|
Unsure
|
2
|
4.5
|
No vision problems
|
5
|
5
|
2
|
4.4
|
Language issue
|
3.5
|
3.5
|
2
|
3.5
|
Un-determined
|
6
|
4.7
|
3.8
|
4
|
Shaking/fall
|
3.8
|
Intermittent with myoclonus at onset of symptoms
|
3.5
|
Unsure
|
Un-determined
|
7
|
5.11
|
3.75
|
5.25
|
Shaking/falls
|
4
|
4.5
|
2
|
Unsure
|
Un-determined
|
8
|
4.1
|
3
|
4
|
Paucity of language and motor development
|
3.5
|
2.5
|
2.5
|
2-3
|
Un-determined
|
Figure 1: Box plots of the age of onset for stages of ambulatory regression.
Figure 1 shows box plots of the age of onset for stages of ambulatory regression. Stages range from gait problems (light blue) down to non-ambulatory status (dark blue). Ages range from roughly 3 years of age to around 6 years of age. Each line corresponds to one of the 8 individual patients.
Figure 2: Dot plot of ambulatory regression.
Figure 2 Dot plot of ambulatory regression showing distribution of the age of onset of gait problems (Gait), onset of walking with aid (Aid), and onset of being non-ambulatory (None).
Neuro-imaging findings:
Abnormal MRI results were observed in all (8/8) cases reviewed. All patients had generalized findings with diffuse cortical and cerebellar atrophy with white matter involvement (see Table 4). Diffuse white matter involvement with T2 hyperintensities were noted in 7/8 cases and periventricular gliosis was reported in 2/8 cases. Thalami were involved in all (8/8) cases with a variable degree of volume loss and gliosis.
Table 4: Neuro-imaging findings
Subject ID
|
Age (years. months)
|
MRI Brain
|
1
|
6.9
|
Significant progressive cerebral and cerebellar atrophy.
Faint regions of T2 signal hyperintensity in the periventricular white matter
Bilateral thalamic atrophy
|
2
|
6.11
|
Generalized parenchymal volume loss and periventricular gliosis.
White matter pallor on T2 Flair images with increased signal intensity in periventricular white matter and posterior limbs of internal capsule
Thalamic atrophy and gliosis
|
3
|
5.5
|
Diffuse supratentorial and infratentorial atrophy
White matter appropriately myelinated
Mild thalamic volume loss with gliosis
|
4
|
5.6
|
Generalized parenchymal volume loss
Periventricular gliosis
Paucity of white matter
Increased T2 signal abnormalities along periventricular white matter
Thalamic volume loss
|
5
|
5
|
Mild to moderate generalized cerebral and cerebellar atrophy associated
Abnormal myelin pallor involving the deep cerebral white matter and corticospinal tracts
Marked thalamic atrophy
|
6
|
4.7
|
Generalized mild cerebral and moderate cerebellar atrophy. Confluent myelin pallor involving the deep cerebral white matter and extending along the corticospinal tracts.
Moderate thalamic atrophy
|
7
|
5.11
|
Mild to moderate generalized cerebral and cerebellar atrophy. Moderate ventriculomegaly
White matter paucity
Mild myelin pallor in the periventricular white matter
|
8
|
4.1
|
Mild to moderate generalized cerebral atrophy with nonobstructive ventricular enlargement
Symmetric myelin pallor involving the deep cerebral white matter with moderate bilateral
thalamic atrophy
|
Electrophysiological findings
Electroencephalographic interictal background activity for all eight patients was abnormal. Interictal background activity showed continuous generalized delta and theta slowing in 37.5% and 62.5% of patients, respectively. In 5 out of 8 patients generalized rhythmic delta activity was seen which often was maximal in the bioccipital or bifrontal regions (Figure 3). Independent multifocal spikes (100% of patients) along with generalized (75% of patients) with posterior maximum epileptiform discharges were seen. During photic stimulation no electroretinogram artifact, photomyoclonus or photoparoxysmal responses were seen in 6 of 8 patients. In one patient photic stimulation triggered a focal motor (myoclonic) seizure with concomitant spike and wave activity seen in the bioccipital regions. One unprovoked seizure was captured, identified by the caregiver, and characterized clinically by a brief head drop (atonic seizure). Ictal EEG showed a generalized spike and wave correlate (Figure 4). One event of non-epileptic staring was seen with no electrographic correlation.
Figure 3: Interictal background showing generalized rhythmic delta activity.
Figure 3 Interictal background showing generalized rhythmic delta activity (between green arrows).
Figure 4: Electro-clinical seizure activity.
Figure 4 Electro-clinical seizure clinically characterized by head drop with a generalized spike and wave ictal correlation.
Neuropsychological assessment results
Assessment of developmental/cognitive abilities was limited for this population given advanced disease progression. Of the 8 children seen, 6 had complete Mullen administrations (participants 3-8). The other two were both evaluated on receptive and expressive language and only one (participant 1) on fine motor abilities. Except for participant 8, all others obtained a standard score at the floor (i.e., lowest score available) of the measure (Standard Score = 49) and participant 8 performed in the exceptionally low range (Early Learning Composite Standard Score = 63). Additionally, participants 1, 2, and 7 were outside of the normative age-range. As such, Developmental Quotients are thought to best capture the participants’ functioning and are presented in Figure 5. Participants 5 and 6 underwent follow-up evaluations with the Mullen demonstrating regression in all domains for both participants (Figure 6). The slope of the regression was most significant in the area of receptive language.
Figure 5: Boxplots of Developmental Quotients from the Mullen for subjects 1-8.
Figure 5 Boxplots of Developmental Quotients from the Mullen for subjects 1-8. The scores represented are the Gross Motor (GM), Fine Motor (FM), Visual Reception (VR), Receptive Language (RL), and Expressive Language (EL) subtests. Subjects from the natural history study (NH) are colored red, and subjects from the gene therapy clinical trials (Clinical) are colored blue.
Figure 6: Dot plot of the Mullen Standard Scores and Developmental Quotients for 2 subjects.
Figure 6 Dot plot of the Mullen Standard Scores and Developmental Quotients for 2 clinical trial subjects from the baseline data (T1) and after the course of 1 year (T2).
With respect to adaptive functioning, there was noted to be significant variability although all but participant 8 was noted to be in the exceptionally low range on the Vineland-3 Adaptive Behavior Composite (Standard Score mean = 51.5 ± 31.5). Composite standard scores for the adaptive measure are presented in Figure 7. Participants in the clinical trial were rated overall at a higher level of functioning than the participants in the natural history portion. Similar to results from the Mullen, follow-up evaluations on the Vineland-3 for participants 1, 2, 5 and 6 also demonstrated parent-reported regression overall (Adaptive Behavior Composite; see Figure 8). Individually, participant 5 had sharp regressions in Communication while the others remained at the floor of the subdomain. Daily Living remained stable for participants 1 and 5 but was noted to regress for participants 2 and 6. Social skills were also relatively stable for participants 1 and 2 but showed a decline in 5 and 6. Further, Motor Skills were already at the floor for participants 1 and 2 but demonstrated rapid regression for participants 5 and 6. A look at the Developmental Quotients for the motor and communication subscales of the Vineland-3 revealed smaller differences between natural history and clinical trial participants. Further, the language scales were rated more highly than motor scales (Figure 9).
Figure 7: Boxplots of Standard Scores from the Vineland-3 for subjects 1-8.
Figure 7 Boxplots of Standard Scores from the Vineland-3 for subjects 1-8. The scores collected are the Adaptive Behavior Composite made up of the other subdomains including Socialization, Daily Living Skills, Communication, and Motor Skills composites. Subjects from the natural history study (NH) are colored red, and subjects from the gene therapy clinical trials (Clinical) are colored blue.
Figure 8: Dot plot of the Vineland-3 composite and domain scores baseline to 1 year follow up.
Figure 8 Dot plot of the Vineland-3 composite and domain scores for 2 natural history subjects (ID: 1 and 2) and 2 clinical trial subjects (ID: 5 and 6) from the baseline data (T1) and after the course of 1 year (T2). Scores represented are the Adaptive Behavior Composite made up of the other subdomains including Socialization, Daily Living Skills, Communication, and Motor Skills composites.
For the clinical trial portion, assessment of health-related quality of life was assessed (participants 5-8). Three participants received the ITQOL (participants 5, 6, and 8). There was significant variability in responses (Figure 10), but parents rated Overall Health as good to excellent. While parents for participants 6 and 8 rated their child’s Change in Health much worse than one year ago, parents of participant 5 rated it to be about the same. Parental Emotional Impact and Family Cohesion also revealed significant variability. The QI-Disability was administered to participants 5 and 7 (Figure 11). The greatest variability was seen in Negative Emotions (e.g., behavioral outbursts, withdrawn behavior), but both families rated generally positive Physical Health, Positive Emotions (e.g., smiling, laughing), Social Interaction, and Leisure Skills.
Figure 9: Boxplots of Developmental Quotients Scores from the Vineland-3 for subjects 1-8.
Figure 9 Boxplots of Developmental Quotients Scores from the Vineland-3 for subjects 1-8. The scores represented are the Gross Motor (GM), Fine Motor (FM), Receptive Language (RL) and Expressive Language (EL) subscales. Subjects from the natural history study (NH) are colored red, and subjects from the gene therapy clinical trials (Clinical) are colored blue.
Figure 10: Boxplots of the ITQOL scores for 3 clinical trial subjects.
Figure 10 Boxplots of the ITQOL scores for 3 clinical trial subjects. Scores represented are Overall Health (OH), Physical Abilities (PA), Growth and Development (GD), Discomfort/Pain (BP), Temperament and Moods (TM), Behavior Overall (GB & GB2), Getting Along with Others (BE & CBE), General Health Perceptions (GH), Change in Health (CH), Parental Impact Emotional (PE), Parental Impact Time (PT), and Family Cohesion (FC).
Figure 11: Boxplots of the QI-Disability scores for 2 clinical trial subjects.
Figure 11 Boxplots of the QI-Disability scores for 2 clinical trial subjects. Scores represented are Physical Health (PH), Positive Emotions (PE), Negative Emotions (NE), Social Interaction (SI), Leisure & Outdoors (LO), Independence (Ind), and Total Score (TS).