In May of 2017, a commission composed of two neurologists (DA, DH) and two neuropsychologists (SM, LM) traveled to the municipality of “Ariguaní” (town of “El Difícil”) in the State of Magdalena, Colombia (Figure 1a) where the affected individuals live. The family's home is far away from the populated area, and there is no road available for motorized vehicles (Figure 1a). Therefore, the trip must be completed by horse or by foot. Once in place, the reconstruction of the pedigree was done by anamnesis following standard procedures. The nuclear family, including the four symptomatic individuals, derives from the mating of two consanguineous first cousins. Endogamous marriage is a common phenomenon in this geographical region because emigration and immigration are limited by the geographical barriers and because the territory is plagued of guerrillas, paramilitary groups and narcotics smugglers. Both parents are unaffected, and the four affected individuals are from a sibship of 12 sibs (Figure 1b). There are twelve descendants in the last generation of the pedigree (Figure 1b).
Neurological and neuropsychological assessment: The anamnesis of symptoms and signs as well as the neurologic examination and other clinical procedures were applied as standardized by the Grupo de Neurociencias de Antioquia form the Medical School, Universidad de Antioquia, Medellín, Colombia and fully described elsewhere28–31 and in the web page: https://web.gna.org.co/en/clinical-studies/
The protocol used for neuropsychological assessment comprises the battery of the CERAD: Mini-Mental State Examination, Semantic Fluency (Animals), CERAD Boston Naming Test, CERAD Word List Learning and delayed recall, CERAD Constructional Praxis Copy and delayed recall.32–34 Episodic memory tests were applied: MIS - Memory Impairment Screening and MCT - Memory Capacity Test.35,36 It is part of the protocol the Rey-Osterrieth Complex Figure Copy and, Phonemic Fluency (FAS) “F”, Wisconsin Card Sorting Test, Trail Making Test -Part A WAIS-III Digit Symbol, Raven’s Progressive Matrices A. The Functional Scales: Barthel, EDG, Katz, Lawton-Brody, Yesavage - Depression Scale, and Memory Disorders Scale, patient, and caregiver. These methods have been described in detail elsewhere.28–31
Case 1
The index case is the older sibling, a 38-year-old illiterate man; his psychomotor development was normal until 5. He debuted with a slow gait with lateral deviation and instability, frequent falls, paresthesias, and occasional finger pain compromise of muscle strength. At 35, he showed calm behavior, regular sleep, and a good appetite and denied memory loss, delusions, hallucinations, disinhibition, depressive episodes, seizures, myoclonus, emotional incontinence, urinary and fecal incontinence. Nevertheless, liquids dysphagia, steppage gait, bilateral foot drop were noticed. The man required assistance for daily life activities. Physical examination showed an alert and collaborative person, facial dyskinesia, mild to moderate dysarthria and intelligible speech, horizontal ocular apraxia, and dysmetria. Both upper and lower extremities showed distal muscle atrophy without pes cavus; muscle strength was 4/5; upper and lower extremities exhibit hyporeflexia and Achillean areflexia, distal hypotonia was also identified, neuropathic gait, and bilateral foot drop. Hoffman, Trommer, and Babinski’s signs were negative. In the upper extremities, a glove spreading hypoalgesia; in lower extremities, bilateral hypoalgesia, starting al L1, were detected (a summary of neurological signs and symptoms for each patient is presented in Table 1). The neuropsychological assessment suggests time-spatial disorientation with impaired cognitive domains and difficulty for complex daily-life activities due to motor symptoms. There were no symptoms or signs of dementia (Table 2).
Table 1
Demographic, epidemiologic, neurological signs, and symptoms differences and similarities found in the clinical examination of the 4 siblings affected with Ataxia with Ocular Apraxia type 1 (AOA1) (APTX, W279*, p.Trp279Ter mutation).
Sign and Symptoms
|
Case 1
|
Case 2
|
Case 3
|
Case 4
|
Complications during pregnancy birth and birth
|
No
|
No
|
No
|
No
|
Age of onset (years)
|
5
|
6
|
11
|
8
|
Instability and frequent falls
|
Yes
|
Yes
|
Yes
|
Yes
|
Paresthesias
|
Yes
|
No
|
No
|
No
|
Physical pain
|
Occasional
|
No
|
No
|
No
|
Dysphagia
|
Yes
|
No
|
No
|
No
|
Behaviour
|
Calm
|
Irritable
|
Calm
|
Aggressive
|
Sleeping patterns and appetite
|
Normal
|
Normal
|
Normal
|
Normal
|
Urinary and fecal incontinence
|
No
|
No
|
No
|
No
|
Memory
|
Normal
|
Normal
|
Normal
|
Normal
|
Delirium, hallucinations or disinhibition
|
None
|
None
|
None
|
None
|
Pseudobulbar affect
|
None
|
None
|
None
|
None
|
Depressive episodes
|
No
|
No
|
No
|
No
|
Seizures and myoclonus
|
No
|
No
|
No
|
No
|
Dysarthria
|
Mild-moderate
|
Mild-moderate
|
Mild
|
N/R
|
Language
|
Understandable
|
Scarce
|
Understandable
|
Understandable
|
Vital Signs
|
Normal
|
Normal
|
Normal
|
Normal
|
Facies
|
Symmetric
|
Symmetric
|
Symmetric
|
Symmetric
|
Fundus examination
|
Normal
|
Normal
|
Normal
|
Normal
|
Telangiectasias
|
No
|
No
|
No
|
No
|
Muscle Tone
|
Lower Limb Hypotonia
|
Atrophy of Gastrocnemius
|
Thenar and gastrocnemius hypotrophy and Hypertrophy of both deltoids
|
Atrophy of Gastrocnemius
|
Ocular-motor Apraxia
|
Yes
|
Yes
|
Yes
|
Yes
|
Strength
|
4/5
|
4/5
|
4/5
|
4/5
|
Generalized Hyporeflexia
|
Yes
|
Yes
|
Yes
|
Yes
|
Hoffman, Trommer and Babinski Reflexes
|
Absent
|
Absent
|
Absent
|
Absent
|
Chorea
|
No
|
No
|
No
|
No
|
Sensitivity
|
Bilateral Glove Hypoalgesia
|
Conserved
|
Bilateral Glove-and-stocking Anesthesia
|
Conserved
|
Dyskinesia
|
Upper limb and Facial
|
Facial
|
None
|
Facial
|
Dysmetria
|
Yes
|
Yes
|
Yes
|
Yes
|
Foot Drop
|
Bilateral
|
Absent
|
Bilateral
|
absent
|
Cavus Foot
|
Absent
|
Absent
|
Absent
|
Absent
|
Table 2
Summary of neuropsychological findings in 4 siblings affected with Ataxia with Ocular Apraxia type 1 (AOA1) (APTX, W279*, p.Trp279Ter mutation).
|
Case 1
|
Case 2
|
Case 3
|
Case 4
|
Age
|
37
|
30
|
23
|
17
|
Mini-Mental State Examination/30
|
17
|
17
|
15
|
19
|
Attention
|
|
|
|
|
Memory
|
|
|
|
|
CERAD Word List Learning /30
|
11
|
13
|
11
|
17
|
CERAD Word List Delayed Recall /10
|
3
|
4
|
4
|
4
|
CERAD Constructional Praxis Delayed Recall/11
|
2
|
0
|
2
|
0
|
Rey-Osterrieth Complex Figure Delayed Recall/36
|
0.5
|
0
|
5.5
|
0
|
MIS Memory Impairment Screen Free Recall /8
|
6
|
6
|
6
|
7
|
Praxis
|
|
|
|
|
CERAD Constructional Praxis Copy /11
|
0
|
0
|
2
|
4
|
Rey-Osterrieth Complex Figure Copy/36
|
0.5
|
0
|
7
|
22
|
Language
|
|
|
|
|
CERAD Boston Naming Test /15
|
6
|
8
|
11
|
8
|
Semantic Fluency (Animals)
|
9
|
5
|
6
|
7
|
Executive Functioning
|
|
|
|
|
Wisconsin Card Sorting Test
|
|
|
|
|
Categories /6
|
1
|
N/A
|
1
|
1
|
Perseverations
|
22
|
N/A
|
28
|
42
|
Phonemic Fluency (FAS) “F”
|
1
|
1
|
N/A
|
N/A
|
Raven’s Progressive Matrices A/12
|
5
|
5
|
8
|
5
|
Functional Scales
|
|
|
|
|
Memory Disorders Scale QP/45 QF/45
|
27/28
|
22/30
|
26/22
|
22/24
|
FAST/16
|
3
|
3
|
3
|
3
|
EDG/7
|
3
|
3
|
3
|
3
|
KATZ/6
|
2
|
2
|
0
|
0
|
BARTHEL/50
|
40
|
40
|
50
|
50
|
Lawton & Brody/8
|
3
|
5
|
5
|
3
|
Yesavage (depression scale)/15
|
11
|
10
|
5
|
11
|
Note: Low performance in some neuropsychological tests might be explained by the poor schooling level and there was not available data for: WAIS-III Digit Symbol Trail Making Test-A (time) MCT Memory Capacity Test -Total Free Recall /32
|
Case 2
Case 2 is the sister of the index case. At age six, she begins to present gait instability and frequent falls. Her menarche was at 12, and she gave birth to a 8-year-old child, born by C-section. At 33-years-old she showed irritable behavior and needed support to develop daily life activities. Physical examination showed steppage and slow gait with lateral deviation, but she was able to walk shortly on flat surfaces without support. Horizontal nystagmus was noticed along with bilateral ocular apraxia, oral dyskinesia, finger-nose dysmetria, mild dysarthria, understandable speech, and low language proficiency. Upper and lower limbs showed distal muscle atrophy, mild atrophy of the gastrocnemius, and muscle strength was 4/5. Brachioradialis hyporeflexia with bilateral bicipital, tricipital, patellar, and Achillean areflexia was noticed. Hoffman, Trommer, and Babinski’s signs were negative, and she did not show sensitivity compromise in the limbs (a summary of neurological signs and symptoms for each patient is presented in Table 1). The neuropsychological assessment suggests time-spatial disorientation with low semantic fluency, problems to nominate, constructional praxis and symptoms of depression. She also had impairment for complex and straightforward daily-life activities due to motor symptoms. She did not show signs or symptoms of dementia. (Table 2).
Case 3
The third affected individual in the family started with difficulty to stand, lateral deviation when walking and frequent falls at eleven years of age. At the age of 26, he showed an ataxic and steppage walking pattern with short steps only in flat sections. He requires assistance with daily living activities and has a mild dysarthria and intelligible speech. Physical examination displays ocular apraxia, horizontal nystagmus, slight dysmetria, pectus excavatum and bradycardia. Hypertrophy of the deltoid muscle was noticed and hypotrophy of the thenar region and gastrocnemius muscle were recorded. Muscle strength was normal in upper limbs but described as a 4/5 in distal lower limbs. Hyporeflexia/areflexia was recorded in upper and lower extremities and she presented a bilateral foot drop. Hoffman, Trommer, and Babinski’s signs were negative. Sensitivity examination showed long superficial glove hypoesthesia and stocking anesthesia (a summary of neurological signs and symptoms for each patient is presented in Table 1). The neuropsychological assessment showed temporospatial disorientation, attention impairment, memory, language, praxis, and executive function. Daily-life activities were impaired due to the motor symptoms but did not have symptoms of dementia (Table 2).
Case 4
The last affected member of this family is a 20-year male, at the time of the interview, with gait instability and frequent falls when walking. This began when he was eight years of age. At the age of 18, difficulty to perform routine life activities and occasional irritable and aggressive episodes were noticed. Physical examination showed that he was able to maintain an unsteady yet unsupported gait. He presented mild dysarthria and understandable language as well as facial dyskinesia without facial muscle weakness. Ocular apraxia, horizontal and vertical nystagmus, mild dysmetria were noticed. The man also had a pectus excavatum. The muscle evaluation showed a normal muscle tone, gastrocnemius muscle atrophy, muscle strength with an overall score of 4/5 in all limbs. His reflexes showed generalized hyporeflexia with Achillean areflexia and normal superficial sensitivity. The neuropsychological assessment showed temporospatial disorientation, semantic and phonologic fluency impairment, episodic memory, constructional apraxia, moderate anomia, and low executive function. He had signs of depression. He was fully independent for complex daily life but did have a multidomain mild cognitive impairment (Table 2).
Whole Exome Capture, Sequencing, And Bioinformatics Analysis
Three methods were applied to DNA quantification, and qualification: (1) DNA purity was checked using the Nanodrop (OD260/280 ratio); (2) DNA degradation and contamination were monitored on 1% agarose gels; (3) DNA concentration was measured using Qubit. DNA samples with OD260/280 ratio between 1.8~2.0 and concentration above 1.0ug were used to prepare sequencing libraries.
Library Preparation for Sequencing
Agilent liquid phase hybridization was applied to enrich whole exons sequenced on an Illumina platform efficiently. Sequencing libraries and capture used Agilent SureSelect Human All ExonV5/V6 (Agilent Technologies, CA, USA) with reagents recommended by the instruction manual and following experimental procedures for optimal results.
Next-Generation Sequencing
Genomic DNA was randomly fragmented to 180-280bp with Covaris cracker, and then DNA fragments were ended polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing. Fragments with specific indexes were hybridized with more than 543,872 biotin-labeled probes after pooling, then magnetic beads with streptomycin were used to capture 334,378 exons from 20,965 genes. After PCR amplification and quality control, libraries were sequenced.
Bioinformatic Analyses
All sequenced data were quality assessed (base quality distribution, nucleotide distribution, and presence of adapters, chimeras, and other contaminants) to identify/remove low-quality data/samples from further analysis. All high-quality data was then be mapped to the human genome assembly using the bwa-mem algorithm.37–39 Aligned files were processed using Genome Analysis Tool Kit (GATK) for base quality recalibration, indel realignments, and duplicate removal.40–42 This was followed by SNP and INDEL discovery and genotyping (plus phasing where applicable) according to GATK Best Practices recommendations.40–42 All variant calls were subject to variant quality score recalibration and filtering to remove low-quality variants. The remaining high-quality variants were annotated for predicted functional consequences using the Voting Report Index, including SIFT, PolyPhen2 HVAR, Mutation Taster, Mutation Assessor, FATHMM, and FATHMM MKL Coding. For example, for a conservative filter, simply keep things that have 0, 1, or maybe two tolerated predictions. A more conservative filter would keep based on 3, 4, or 5 Damaging predictions. Many variants do not have five algorithms with non-missing values (see Supplementary Material). Updated annotations from the NCBI, the 1000 genome project, were used to evaluate the novelty and rareness of variants. Methods for the selection of variants have been widely described elsewhere.42–48
Tridimensional Protein Reconstruction and Functional Effects by in silico Analysis
We use the significant improvement in the accuracy of protein structure prediction recently implemented in AlphaFold50 that incorporates novel neural network architectures and training procedures based on the evolutionary, physical, and geometric constraints of protein structures. Tridimensional protein structure reconstruction with AlphaFold is vastly more accurate than those obtained by competing methods, i.e., median backbone accuracy, highly accurate side chains reconstruction, accurate domains, and domain-packing prediction, and precise, per-residue estimates of its reliability. Code in Phyton is available upon request.