Hereditary Cerebellar Ataxias in the Era of Next Generation Sequencing

Mestrado Integrado em Medicina

Hereditary Cerebellar Ataxias in the Era of Next Generation

Sequencing

Duarte Foja Ferreira Pinto Ceia

M

2022

Hereditary Cerebellar Ataxias in the Era of Next Generation Sequencing

Dissertação de candidatura ao grau de Mestre em Medicina, submetida ao Instituto de Ciências Biomédicas Abel Salazar – Universidade do Porto

Estudante: Duarte Foja Ferreira Pinto Ceia

Aluno do 6º ano de Mestrado Integrado em Medicina Endereço de correio eletrónico: duarteceia@gmail.com

Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto Endereço: Rua de Jorge Viterbo Ferreira nº228, 4050-313 Porto

Orientador: Joana Catarina Damásio Correia dos Santos, MD

Assistente Graduada em Neurologia, Centro Hospitalar Universitário do Porto

Afiliação: Serviço de Neurologia, Hospital de Santo António, Centro Hospitalar Universitário do Porto

Endereço: Largo Prof Abel Salazar, 4099-001 Porto

Coorientador: Manuel Jorge Maia Pereira Correia, MD PhD

Professor Catedrático, Convidado com agregação do Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto,

Assistente Graduado Sénior de Neurologia no Centro Hospitalar Universitário do Porto

Afiliação: Serviço de Neurologia, Hospital de Santo António, Centro Hospitalar Universitário do Porto

Endereço: Largo Prof Abel Salazar, 4099-001 Porto

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A GRADECIMENTOS

À Drª Joana Damásio e ao Professor Manuel Correia, pela disponibilidade e por todos os ensinamentos ao longo do processo de redação do documento.

Aos meus pais e ao meu irmão, por todo o apoio e por sempre acreditarem em mim.

Aos meus amigos e colegas de curso, pelas memórias e pelo auxílio ao longo destes 6 anos.

v

A BSTRACT

Background: Hereditary ataxias are a group of neurological disorders due to degeneration of the cerebellum/ cerebellar pathways, dorsal columns or sensitive nerves. In recent years its diagnosis has been revolutionized by Next Generation Sequencing techniques, allowing the simultaneous study of several genes. Notwithstanding its importance, the identification of variants of uncertain significance has increased, posing an additional challenge.

Objective: To describe a patient with hereditary cerebellar ataxia and variants in SACS and APTX genes identified by Next Generation Sequencing. To analyse the phenotype in correlation with the identified variants and review the phenotype of SACS and APTX genes. To perform a review on patients with neurological disorders bearing variants in two or more genes.

Methods: A structured protocol was developed for data collection and a retrospective review of the files was performed, completed with an in-person observation. A PubMed review was performed for A) genotype-phenotype associated with pathogenic variants in SACS and APTX;

B) patients with pathogenic variants in two or more genes associated with neurological disorders.

Results: The patient is a 51 years old female, born from consanguineous parents, with progressive gait difficulties since the age of 30 who, over time, developed a cerebellar syndrome, pyramidal signs, polyneuropathy, limb oedema and oculomotor apraxia. She had a history of

“meningitis” at the age of 2 years old, and epilepsy from the age of 8 until 13. MRI disclosed cerebellar and pons atrophy, thin corpus callosum, hyperintensity of the pons and middle cerebellar peduncle and spinal cord atrophy. On Next Generation Sequencing panel it was identified: c.4521_4522del(p.(Asn1508Serfs*32)), a likely pathogenic variant, in homozygosity in SACS; and c.837G>A(p(Trp279)), a known pathogenic variant, and c.762G>A(p.(=)), a variant of uncertain significance, in compound heterozygosity in APTX.

Discussion: This patient presents a complex phenotype. Age of onset, epilepsy and ataxia together with pyramidal signs are consistent with pathogenic variants in SACS, while oculomotor apraxia and marked limb oedema (as well as ataxia) are more frequently identified in pathogenic variants in APTX, but with such a late onset being very uncommon. From a genetic point of view, the pathogenicity of the SACS variant has been demonstrated, while one of the variants in APTX was classified as variant of uncertain significance. We hypothesize there could be a modulation

vi of the SACS phenotype by the APTX variants, but additional studies would be needed to better characterize this.

Keywords:

“Hereditary Cerebellar Ataxia”; “Next Generation Sequencing”

vii

R ESUMO

Contexto: As ataxias hereditárias são um grupo de distúrbios neurológicos que resultam da degeneração do cerebelo/ vias cerebelosas, dos cordões posteriores da medula ou dos nervos sensitivos. Recentemente, o seu diagnóstico foi revolucionado pelas técnicas de Sequenciação de Nova Geração, que permitiram estudar simultaneamente vários genes. Apesar da sua importância, têm sido identificadas cada vez mais variantes de significado indeterminado cuja interpretação tem sido desafiante.

Objetivo: Descrever uma doente com ataxia cerebelosa hereditária e variantes nos genes SACS e APTX que foram identificadas recorrendo a técnicas de Sequenciação de Nova Geração.

Analisar o fenótipo e correlacioná-lo com as variantes identificadas. Rever o fenótipo associado a variantes patogénicas nos genes SACS e APTX. Fazer uma revisão em doentes com doenças neurológicas e variantes em ≥2 genes.

Métodos: O ficheiro clínico foi retrospetivamente analisado tendo em conta um protocolo estruturado que foi elaborado para colher informação do mesmo. Posteriormente, fez-se uma avaliação presencial da doente. Foi feita uma revisão no PubMed sobre A) genótipo e fenótipo associados a mutações patogénicas nos genes SACS e APTX; B) doentes com distúrbios neurológicos e variantes patogénicas em ≥2 genes.

Resultados: Doente do sexo feminino, com 52 anos, filha de pais consanguíneos, com dificuldade na marcha progressivamente maior desde os 30 anos e que desenvolveu síndrome cerebelosa, sinais piramidais, polineuropatia, edema dos membros inferiores e apraxia oculomotora. Tinha antecedentes pessoais de “meningite” aos 2 anos de idade, e epilepsia entre os 8 e os 13. A RMN mostrou atrofia do cerebelo e da ponte, um corpo caloso fino, hiperintensidades na ponte e no pedúnculo cerebeloso médio e atrofia da espinal medula. No painel de Sequenciação de Nova Geração foram identificadas:

c.4521_4522del(p.(Asn1508Serfs*32)), uma variante provavelmente patogénica, em homozigotia no gene SACS; c.837G>A(p(Trp279)), uma variante patogénica conhecida, e c.762G>A(p.(=)), uma variante de significado indeterminado, em heterozigotia composta no gene APTX.

viii Discussão: Esta doente apresenta-se com um fenótipo complexo. A idade de início, a epilepsia e a ataxia, juntamente com os sinais piramidais, são consistentes com variantes patogénicas no SACS, enquanto a apraxia oculomotora e o edema dos membros inferiores (assim como a ataxia) são mais frequentemente identificados nas variantes patogénicas do gene APTX, embora seja raro um início tão tardio. De um ponto de vista genético, a patogenicidade da variante do SACS foi demonstrada, enquanto uma das variantes do gene APTX foi classificada como variante de significado indeterminado. Nós pensamos que variantes no gene APTX poderão alterar o fenótipo associado ao SACS, mas estudos adicionais serão necessários para melhor caracterizar isto.

Palavras-chave:

“Ataxias Cerebelares Hereditárias”; “Sequenciação de Nova Geração”

ix

A BBREVIATION L IST

ACMG – American College of Genetics and Genomics AD – Autosomal Dominant

AOA – Ataxia with Oculomotor Apraxia

AOA1 – Ataxia with Oculomotor Apraxia type 1 AR – Autosomal Recessive

ARSACS – Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay BBS - Bardet-Bield Syndrome

CHUPorto - Centro Hospitalar Universitário do Porto DRPLA - Dentatorubro-Pallidoluysian Atrophy DSBs - Double-Strand Breaks

EEG – Electroencephalogram EMG – Electromyography FA – Friedreich Ataxia HIT - Histidine Triad

MJD – Machado-Joseph Disease MRC – Medical Research Council NGS – Next Generation Sequencing OCT - Optical Coherence Tomography PARP-1 - Poly-ADP Ribose Polymerase 1 SCA2 – Spinocerebellar Ataxia type 2 SSBs - Single-Strand Breaks

TRPS type 1 - Trichorhinophalangeal Syndrome type 1 VUS - Variant of Uncertain Significance

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I NDEX

Agradecimentos ...iv

Abstract ... v

Resumo ... vii

Abbreviation List ... ix

Index ... x

Table List ... xi

Figure List ... xii

Attachment List ... xiii

Introduction ... 1

Objectives ... 3

Materials and Methods ... 4

A. Patient ... 4

B. Literature review on ARSACS and AOA1 ... 4

RESULTS ... 6

Case Presentation ... 6

Gestation, Childhood and Early Adulthood - Social Perspective ... 6

Medical History ... 6

Family History ... 8

Neurological Exam (51 years old) ... 8

Diagnostic Work-up ... 9

Literature Review ... 11

Ataxia with Oculomotor Apraxia type 1 (AOA1) ... 11

Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS) ... 13

Patients with Neurological Disorders and Pathogenic Variants in Two or More Genes ... 15

Discussion ... 18

Conclusion ... 20

Tables ... 21

Figures ... 39

Attachments ... 43

Bibliography ... 47

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T ABLE L IST

Table I - Patients with neurological disorders and pathogenic variants in two or more genes

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F IGURE L IST

Figure 1 - Genogram of the patient’s family Figure 2 – First neuroaxis MRI (2009) Figure 3 – Second neuroaxis MRI (2016)

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A TTACHMENT L IST

Attachment 1 – Structured protocol for data collection

1

I NTRODUCTION

Ataxia is described as an impairment in coordination of voluntary movements due to pathology of the cerebellum, its afferent/ efferent pathways or the proprioceptive system. Appendicular ataxia results of cerebellar hemisphere’s lesions and consists of intentional tremor and dysdiadochokinesia, while stance and gait ataxia appears when the vermis is affected. Other symptoms also present in cerebellar ataxia are dysarthria and eye movement abnormalities¹. The most frequent causes of ataxia are acquired¹ (e.g. stroke, cerebellar tumors, multiple sclerosis), but a genetic etiology should be suspected whenever there’s family history of a similar disorder or consanguinity. Overall autosomal recessive (AR) ataxias are more prevalent (3,3/100000 inhabitants) than autosomal dominant (AD) forms (2,7/100000 inhabitants)². Worldwide, the most prevalent AR ataxias are Friedreich ataxia (FA) and ataxia-telangiectasia, while for AD ataxias is Machado-Joseph disease (MJD), followed by spinocerebellar ataxia type 2 (SCA2)². Some authors suggest that the underlying reason for MJD being the most prevalent ataxia is related with the Portuguese overseas expansion in the XV century, after a founder effect occurred in the Azores Islands³. The countries with the highest prevalence of AR ataxias are Spain and France, and the ones with the highest prevalence of AD ataxias are Portugal, Japan and Norway².

In Portugal, AD ataxias (5,6/100000 inhabitants) are more prevalent than AR forms (3,3/100000 inhabitants). The most prevalent AD ataxias are MJD and dentatorubro-pallidoluysian atrophy (DRPLA), with AR ataxias being FA and ataxia with oculomotor apraxia (AOA)⁴. MJD typical age of onset is in the 4^th decade of life, with the patients presenting slowly progressive cerebellar ataxia in association with pyramidal signs, neuropathy, and, less frequently, movement disorders^3,5. DRPLA has a very large spectrum of ages of onset and clinical presentations, with the youngest patients presenting progressive myoclonic epilepsy in association with ataxia, and the oldest ones chorea in association with ataxia⁶. FA is characterized by sensitive ataxia, neuropathy and extra-neurological symptoms, with typical onset between the ages of 10 and 16⁷. AOA has oculomotor apraxia as a distinctive feature, with four types described thus far.

AOA1, caused by pathogenic variants in APTX, is characterized by childhood-onset slowly progressive cerebellar ataxia followed by oculomotor apraxia and peripheral neuropathy⁸. A rare cause of ataxia is autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), initially described in the Charlevoix and Saguenay regions of Canada. Clinically, it manifests with

2 childhood-onset gait and postural imbalance, followed by lower limb spasticity and later peripheral neuropathy. A very characteristic finding is the presence of yellow streaks of hypermyelinated retinal fibers on fundoscopy⁹. These last two forms of genetic ataxias will be the main focus of this thesis.

Genetic diagnosis has greatly evolved in the past decade, with the introduction of next- generation sequencing (NGS) in clinical practice. In hereditary ataxias, NGS has significantly increased the diagnostic rate¹⁰. NGS technique stands on binding single stranded genetic material to a solid surface coated with adapter oligonucleotides. These DNA molecules are then amplified and identified through a variety of methods, depending on the equipment. When compared to Sanger sequencing, NGS is both more sensitive and time-saving, since it reads many genes at the same time. The drawbacks are related with logistic requirements, high intrinsic error rates when sequencing large amounts of genome (due to the amplification process)¹¹, and identification of novel variants that may or may not be associated with the disease being studied or with other health conditions the patients might have. There is no consensus on the best approach to have with the information obtained via these incidental findings, and the protocol varies from laboratory to laboratory^12,13. With regards to novel variants, the American College of Genetics and Genomics (ACMG) guidelines on the interpretation of sequence variants classify them in five different tiers: “pathogenic”, “likely pathogenic”, “uncertain significance”, “likely benign” and “benign”¹⁴. Variants of uncertain significance (VUS) may be identified in up to 40%

in multiple-gene panels¹⁵, posing an additional challenge for the scientific community.

Bioinformatic tools are very helpful^12,13 but may not provide a definite answer, with functional studies being needed.

In this thesis we will discuss the challenges of NGS, through the analysis of a case report.

3

O BJECTIVES

The defined objectives for this thesis were:

 To describe a patient with hereditary cerebellar ataxia who bears variants in SACS and APTX genes, identified by NGS;

 To analyze the patient’s phenotype, in correlation with the identified variants;

 To review SACS and APTX phenotypes;

 To perform a review on patients with neurological disorders bearing variants in two or more genes;

 To discuss the challenges posed by new genetic technologies.

4

M ATERIALS AND M ETHODS A. Patient

A retrospective study of a female patient with early-adulthood onset ataxia, presumably from a genetic cause, was conducted. The study was approved by the Ethics Committee of Centro Hospitalar Universitário do Porto (CHUPorto), and informed consent was obtained. A structured protocol (attachment 1) was elaborated for the file’s retrospective review. The following data were collected:

 Age at first symptom;

 Type of first symptoms;

 Age at onset of the different symptoms/ signs: dysarthria, nystagmus, eye movement abnormalities, hypotonia, appendicular dysmetria, gait abnormalities, postural instability, pyramidal signs, sensitive changes, chorea, dystonia, cognitive decline and non-neurological symptoms;

 Symptom characterization and progression;

 Age of loss of independent gait and ambulation;

 Age at diagnosis;

 Family history;

 Brain MRI;

 EMG;

 Neuropsychological examination;

 Genetic testing.

A complete neurological exam was performed at the in-person evaluation.

B. Literature review on ARSACS and AOA1

A literature review was performed on pathogenic variants in SACS (corresponding to ARSACS phenotype) and APTX (corresponding to AOA1). A second review was made on case reports of patients with neurological disease and pathogenic variants in two or more genes. The searches were conducted on PubMed (https://pubmed.ncbi.nlm.nih.gov/) and took into consideration literature published from January of 2000 until October of 2021. This period was selected since the SACS gene was described in January of 2000. The search terms were as following:

5

 “Spastic ataxia” OR “ARSACS” OR “autosomal recessive spastic ataxia of Charlevoix- Saguenay” OR (“ataxia” AND “SACS”);

 “Ataxia with oculomotor apraxia type 1” OR “AOA1” OR (“ataxia” AND “APTX”);

 Review on patients with pathogenic variants in two or more genes associated with neurological diseases - the following MeSH terms were used: “Humans” AND “Nervous System Diseases/genetics” AND “High-Throughput Nucleotide Sequencing”, which were associated with the non-MeSH terms “(two or more mutations OR two or more variations OR two or more variants)” AND “(association OR causative OR associated)”. A filter was then applied so only case reports were taken into consideration.

6

RESULTS

Case Presentation

The patient reported is a 51 years-old female, from the north of Portugal and retired by disability since the age of 36.

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Both gestation and delivery were within the expected, as well as psychomotor developmental milestones. She had a good relationship with other children and played well with them. She was able to walk, run and move just like her peers. She failed to pass two years at elementary school and dropped out of the 4^th grade. Her teenage years were apparently normal: she learned to play the piano and used to do artcraft “renda de bilros”. During her adult life, she worked as a waitress from the age of 17 until she was 30. She got married, had a child who died at the age of 4 years old, of meningitis. After that she got divorced and never got another employment.

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At the age of 2, she had an episode said to be “meningitis” (no further details available). From the age of 8 to 13 years old, she had epilepsy (described as tonic-clonic seizures), treated with valproic acid, and was later diagnosed with Bernard-Soulier syndrome. When she was 30 years- old, the first motor symptoms appeared, characterized by gait difficulties. She initially felt her left leg was stuck, feeling this later in her right leg as well. She had difficulties lifting her feet, with progressive worsening over time. Difficulty using her hands was noticed when she was 32 as she frequently dropped objects, and a scanning speech also appeared around that age. She started using crutches when she was 35 years-old, and became wheelchair bound seven years later.

She had been observed at another hospital for several years (complete registries not available) and on a neuropsychological evaluation at age 37 there were mild deficits in attention, language, memory, visual perception, visuoconstruction and executive functions – with the patient presenting high levels of anxiety and depression at that time.

7 When first seen at the neurology outpatient clinic of CHUPorto, aged 42, she had scanning dysarthria, gaze evoked nystagmus and fragmented saccadic eye movements. Spastic paraplegia grade 2 (MRC scale) with brisk deep tendon reflexes, ankle clonus and bilateral Babinski sign were present. She had stocking glove hypoesthesia with normal proprioception and vibration sense. There was bilateral upper limbs dysmetria, while lower limbs coordination could not be evaluated due to motor deficit. She was only able to stand with bilateral help, and gait was also not possible (even with bilateral help).

Difficulties in saccadic eye movements, resembling oculomotor apraxia were noticed by the age of 45 years, at the same time of mild dystonic posturing of the upper limbs. Spasticity has progressively decreased over time, with hypotonia registered since the age of 46. Marked lower limb oedema has been present since the age of 50.

In her last medical appointment, she was doing the following medication:

 Riluzole 50 mg 2x per day

 Sertraline 50 mg 1x per day

 Baclofen 30 mg 3x per day

 Diazepam 5 mg 2x per day

 Mirtazapine 15 mg

 Tizanidine 4 mg 3x per day

 Gabapentin 600mg 1x per day

 Tramadol 100 mg 3x per day

 Senna 12 mg 1x per day

 Lactulose

 Bisacodyl 5 mg 1x per day

 Zolpidem 10 mg 1x per day

 Trazodone 150 mg

8

 Paracetamol 1 g up to 3 times per day when needed

 Metoclopramide when needed

F

H

Family tree is displayed in figure 1. Her family was from a small village in the north of Portugal, and her parents were 1^st degree cousins – her maternal grandfather and her paternal grandmother were brothers. Her father died with 70 years of age due to a stroke, and her mother had bipolar disorder and committed suicide at the age of 55. She has 3 healthy half- brothers from her father’s side. There are no other family members diagnosed with a genetic disease.

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(51

)

At the in-person observation the patient presented a scanning dysarthria. There were no language or memory deficits, neither agnosia nor limb-kinetic apraxia. Visual acuity and fields were intact. Pursuit eye movements were fragmented and incomplete on vertical and horizontal planes, which was partially reversible with oculocephalic maneuver. Gaze evoked sustained horizontal nystagmus was present. On saccadic eye movement she had cephalic movement – oculomotor apraxia. Pinprick sensation on the face was normal, as well as masseter and temporal muscle strength. Facial mimic was also normal, as well as her hearing. Trapezium and sternocleidomastoid muscle strength was maintained on both sides. On tongue protrusion there was no lateralization.

She had interosseous muscle wasting and tone was diminished both on upper and lower limbs.

Hand extension was bilaterally grade 4 MRC. Biceps, triceps and brachioradialis hyperreflexia was present. She had pes cavus and marked lower limb oedema. She was paraplegic, patellar and ankle hyperreflexia was present, as well as unsustainable ankle clonus. Plantar reflex was bilaterally indifferent. She had stocking glove hypoesthesia, vibration was preserved on the fingers and reduced on the halluces. There was mild dystonic posturing of the hands. On finger- to-nose and finger-to-finger tests there was dysmetria, and dysdiadochokinesia was also identified. Lower limb coordination could not be evaluated due to motor inability. She could only remain in orthostatism with bilateral help and gait was not possible.

9

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Over the years, an extensive investigation was conducted. We highlight two neuroaxis MRIs, two electromyographies (EMG), one electroencephalogram (EEG) and various genetic tests.

The first neuroaxis MRI was done when she was 38 years old (figure 2). It showed, at a supratentorial level, bilateral hemisphere atrophy, ventricle enlargement, a thin corpus callosum and hyperintensity of the posterior peri-ventricular white matter and of the posterior arms of the internal capsule. In the posterior fossa there was superior vermis atrophy and hyperintensity of the transverse pathways of the pons and of the median brainstem raphe. In the spinal cord, there was generalized atrophy, more pronounced in the dorsal segments, and two discal hernias (one at C5-C6 and the other at L5-S1) with no radicular compromise.

A second brain MRI was conducted seven years later (figure 3). At a supratentorial level, it was similar to the previous one. Cerebellar atrophy was more pronounced in the superior vermis, and both cerebellar hemispheres also displayed atrophy. In the brainstem, the changes were like the ones previously described, but there was also hyperintensity of the middle cerebellar peduncles.

The first EMG (36 years old) described a sensorimotor polyneuropathy. A second EMG was performed when she was 45 years old, again revealing axonal sensorimotor polyneuropathy with a distal-proximal gradient and chronic probably neurogenic muscle wasting. EEG (aged 38) was normal.

The first genetic approach consisted of a NGS panel of 4 genes (SPG7, SPG11, SPG15, SPG21) for autosomal recessive spastic paraplegias, which was negative. Afterwards FXN gene was also studied, again negative. Later, leukocyte galactocerebrosidase activity was determined to search for Krabbe’s disease. The test showed a slightly reduced enzyme activity, but considered normal by the laboratory, excluding Krabbe’s disease. An extensive laboratory workup was performed over the years of which we highlight: albumin in lower range (3.59 g/dl for a normal of 3.50-5.0 g/dl), normal cholesterol and chronic thrombocytopenia (57*10³ /L).

NGS panel for autosomal recessive ataxias (117 genes) was performed when she was 45 years old. A homozygous variant in the SACS c.4521_4522del(p.(Asn1508Serfs*32)) in exon 10 was identified. This variant had not been previously described, but as it produced a truncated protein

10 with probable loss of function, it was classified as likely pathogenic (ACMG classification).

Variants in compound heterozygosity were also identified in APTX gene: c.837G>A(p(Trp279)) in exon 8 - already reported as pathogenic; and a rare variant, c.762G>A(p.(=)), in exon 7 - this variant had not been previously reported, bioinformatic analysis revealed it would change the splicing of the protein being classified as VUS.

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Literature Review

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1 (AOA1)

The search resulted in 193 results. Of these results, only 74 articles were found to be related to AOA1 and the subjects discussed in this work. Ten were chosen for a complete reading, selected according to their relevancy to the topic (protein function, pathophysiology, epidemiology, clinical features, diagnostic exams and genotype-phenotype correlations), title, abstract, journal, year of publication, accessibility to the full article and language (only articles in Portuguese and English).

Ataxia with oculomotor apraxia type 1 is an AR ataxia caused by pathogenic variants in APTX gene, which encodes for the protein aprataxin⁸. The function of aprataxin is related to the repair of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). AOA1 is associated with loss of function variants in APTX, producing structurally deficient aprataxin proteins. These faulty aprataxin proteins fail to recruit other proteins that also participate in DNA SSB repair, like XRCC1 and poly-ADP ribose polymerase 1 (PARP-1), and DSB repair, such as XRCC4^16,17. Due to this, affected cells become hypersensitive to oxidative DNA damage. This is most troublesome in the brain, where energy production and oxygen consumption is high, leading to the formation of free radicals and higher oxidative stress in comparison to other tissues. This explains the fact that AOA1 has mostly neurological symptoms¹⁶. More recent studies suggest that aprataxin is also associated with mtDNA repair, explaining the fact that coenzyme Q10 levels are reduced in some patients¹⁸.

Depending on the region of the world, AOA is the second or third most prevalent cause of AR cerebellar ataxia⁸. In Portugal, it ranks second after Friedreich ataxia⁴, being also prevalent in Japan⁸. In Portugal, the most common pathogenic variant is W279X, a nonsense variant, and in Japan it is the frameshift variant 689insT. Both are the most widespread variants in their countries due to a founder effect.

Clinically, AOA1 is characterized by cerebellar ataxia, oculomotor apraxia and peripheral neuropathy. It can also be associated with chorea, dystonia, cognitive impairment, hypoalbuminemia and hypercholesterolemia⁸. It has a mean onset age of 4,3 years, but it can occur later in life when it is associated with missense mutation ^8,19. There have been reported cases of disease onset as late as 40 years²⁰.Cerebellar ataxia is usually the first symptom, associated with gait imbalance and later dysarthria and upper-limb dysmetria with intentional tremor⁸. Oculomotor apraxia emerges after, being the most characteristic feature of this disease

12 but not being always present (only in 86% of patients)^8,21. When asked to perform a saccadic eye movement, patients will turn their head first, requiring a brief pause before the eyes follow it.

With disease progression, eye movements become impossible without head movement and patients may also have exaggerated blinking. In extremely advanced disease, these findings disappear because a progressive external ophthalmoplegia dominates the clinical picture⁸. The last of the three main features that usually emerges is peripheral neuropathy. It starts with areflexia that slowly progresses into loss of independent walking during adolescence. This progression of the neuropathy along with the gait imbalance forces the patient to use a wheelchair. Although neuropathy may be subclinical in early phases, 100% of patients with AOA1 have clinically relevant peripheral neuropathy by the time they reach adulthood^8,21. Late and slow onset of peripheral neuropathy has also been described. Due to this, patients develop muscular atrophy and around 30% have pes cavus. In later stages of the disease, this dominates the clinical picture because patients eventually become tetraplegic⁸. Choreic movements are present in about 80% of patients at disease onset. However, over time this symptom disappears in approximately 50% of affected patients. Because of this, chorea and cerebellar ataxia dominate the clinical picture in early phases of the disease^8,21. One mutation (A198V) has been associated with a phenotype whose main feature is chorea²¹. Another extra-pyramidal symptom that may appear in up to 50% of patients with AOA1 is upper-limb dystonia⁸.

When the disease was first described, it was thought that mental retardation was only present in Japanese patients with AOA1^22,23. However, recent studies suggest that cognitive impairment of various degrees can be present in patients of all ethnic groups. The clinical heterogeneity of this symptom implies that, in certain patients, signs of cognitive impairment are only found after neuropsychological testing. This cognitive impairment consists of a subcortical syndrome^8,21. Hypoalbuminemia occurs more than 10-15 years after disease onset, being present in around 83% of patients⁸. In approximately 46% of patients, hypoalbuminemia leads to limb oedema²¹. Hypercholesterolemia occurs more than 10-15 years after disease onset. It is present in around 68% of patients. The rise in total cholesterol levels correlates with the decrease of albumin levels^8,21. MRI usually discloses cerebellar atrophy that is more severe in the vermis, leaving the brain hemispheres and the brain stem intact (however, in rare cases, brain stem atrophy has also been reported)⁸.

Some degree of genotype-phenotype correlation is present in AOA1. As described, some missense mutations are associated with later onset and slower disease progression. The variability in clinical severity of the various missense mutations can be justified with their

13 position in relation to the histidine triad (HIT) domain of the APTX gene¹⁹. There is also one mutation (A198V) that, if present, correlates with a phenotype of severe and persistent chorea²¹. In one Tunisian family, a complete deletion of the APTX gene was reported but it was not associated with a more severe phenotype. As such, the classical phenotype of AOA1 is associated with the total loss of function of the aprataxin protein²⁴.

A

R

S

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C

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(ARSACS)

The search disclosed 389 results. Of these results, only 152 articles were found to be related to ARSACS. Thirteen articles were then selected for a complete reading according to their relevancy to the topic (pathophysiology, epidemiology, clinical features, diagnostic exams and genotype- phenotype correlations in ARSACS), title, abstract, journal, year of publication, accessibility to the full article and language (only articles in Portuguese and English).

Autosomal recessive spastic ataxia of Charlevoix-Saguenay is an AR hereditary ataxia caused by loss of function pathogenic variants in SACS gene, which encodes the protein sacsin⁹. The exact function of sacsin is unknown, but several lines of investigation suggest that sacsin function loss leads to cytoskeletal and mitochondrial abnormalities, so it’s likely these defects are related to the pathogenesis of the disease^9,25. Loss of functional sacsin has been found to interrupt intermediate filament organization, leading to vimentin cytoskeleton collapse and organelle distribution abnormalities. The latter might be also related to the mitochondrial deficits recognized in affected cells²⁵. Morphologically altered mitochondria have been identified in affected cells, being speculated that it might be related to deficits in mitochondrial fission. Apart from that, the above-mentioned organelle distribution abnormalities cause accumulation of these faulty mitochondria in the proximal dendrites of neurons, explaining the Purkinje cell loss that is seen in patients with ARSACS^26,27.

This disease is most common in the Saguenay-Lac-St-Jean region of Quebec, in the northeast region of Quebec City in Canada⁹. This is due to a founder effect that occurred in the region during the XIX century, when a group of individuals carrying a mutation in SACS came from Europe and settled there²⁸. This founding mutation was the 6594delT, which is still the most prevalent mutation in the region and worldwide. In Quebec, the estimated carrier frequency of variants in the SACS gene is 1:21 and the birth incidence of ARSACS was 1:1932, but it has decreased in recent years due to voluntary screening of the population⁹.

14 Nowadays, with the development of neurogenetics and NGS techniques, patients with ARSACS have been reported in several countries^9,28. In the past, this disease was considered rare outside of the Quebec region^29,30, but studies have shown that the relative frequency of SACS mutations in patients with early-onset spastic ataxia could reach 37% in some populations (e.g.

Netherlands)^30,31. If we add this to the fact that ARSACS has a very variable clinical presentation, the true prevalence of the disease is likely higher than anticipated³⁰.

ARSACS phenotype is very variable. The 3 main clinical features of this disease are slowly progressive cerebellar ataxia, lower-limb spasticity and peripheral neuropathy⁹. Even though only 78% of patients develop this classic triad, there have been no reported patients with ARSACS and none of these 3 symptoms³². Other symptoms include dysarthria, dysphagia, dystonia, nystagmus, hearing loss, myoclonic epilepsy, cognitive impairment and urinary disturbances. The average life expectancy of patients with ARSACS is 51 years⁹. The onset is usually before the age of 10 years of age⁹, but the classic triad only develops during teenage years, being rare at disease onset. Cerebellar ataxia is usually the first symptom, present in about 78.9%, followed by lower-limb spasticity, in 78.1% of patients^9,33. The last symptom of the triad to manifest is peripheral neuropathy, sensitive and motor, identified in 73.7% of patients. Most frequently it is of demyelinating type, progressing to axonal later. The second most common phenotype of ARSACS is a pronounced peripheral neuropathy starting at an early age^9,33. The remaining symptoms may differ from patient to patient and usually have a slower progression.

Dysarthria is present in 74% of patients, usually starting during late childhood. Nystagmus is also described in 74% of patients. Urinary disturbances (34%) include urgency and incontinence and are usually due to pyramidal tract disease. About 74% of patients have nystagmus. The rest of the symptoms are much less frequent (<25% of the patients develop them)⁹. Mitral valve prolapse is also associated with ARSACS, but it has only been reported in 1 individual that wasn’t French-Canadian⁹.

ARSACS is associated with a large variety of brain MRI findings. The most sensitive and specific is the presence of parallel linear hypointense stripes in the paramedian region of the pons on T2 and T2-FLAIR sequences³³. Other findings include lateral pons hyperintensities (on T2 sequence) that merge with thickened middle cerebellar peduncles, superior vermis or global cerebellar atrophy and hyperintense rims around the thalami^9,34. Some patients might have a normal brain MRI. However, in most of these patients, a retrospective inspection of the images after ARSACS diagnosis revealed the presence of linear hypointensities in the pons⁹.

15 Another specific finding in ARSACS is the presence of hypermyelinated peripapillary retinal fibers on fundoscopy or on optical coherence tomography (OCT)³⁴ not associated with vision abnormalities⁹. Even though the frequency of this finding is usually low (in about 26.3%³³), a study of Brazilian patients revealed a prevalence of almost 100%³⁴. The fact that vision may remain unchanged throughout the whole duration of the disease might contribute to the underidentification, since patients are not subject to routine ophtamological exams³³.

Many patients lack one or more of the three main clinical features of this disease. And the onset has been reported to range from 3 months to >40 years^30,33. However, genotype-phenotype correlation is poor, and no single mutation has been able to justify a specific clinical presentation. The only genotype-phenotype correlation to date shows that, using computational algorithms, the pathogenicity score of SACS gene mutations is directly linked with disease severity. Mutations with a high pathogenicity score are associated with earlier onset and more severe disease³³.

Due to the high prevalence of carriers of variants in the SACS gene³¹, it is common to identify VUS in this gene when using NGS techniques³⁵. When this happens, screening for such variants in the other family members is crucial to determine the pathogenicity of the mutation³⁶. Using computational algorithms to determine its pathogenicity score is also helpful. It is important to mention that almost every new reported case of ARSACS outside of Quebec are associated with the discovery of a new pathogenic variant³³.

P

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NGS has revolutionized the diagnosis of hereditary neurological disorders, with testing of multiple genes at the same time¹³ and expansion of phenotype-genotype classical correlations.

The capability of large-scale genetic testing came with the detection of novel variants, some of unknown significance, being crucial to evaluate it according to its pathogenicity³⁷.

This search resulted in 192 results. Of these results, only 25 articles were chosen. They were filtered according to their relevancy to the topic, title, abstract, journal, year of publication,

16 accessibility to the full article and language (only articles in Portuguese and English). The results are detailed in Table I.

Briefly, in the articles by Hannah-Shmouni, F., et al³⁸, Hata, Y., et al³⁹, Wayhelova, M., et al⁴⁰, Wiedemann, A., et al⁴¹, Marttila, M., et al⁴², Dangiolo, S.B., et al⁴³, Moteki, H., et al⁴⁴ and Nectoux, J., et al⁴⁵, the possibility of diagnosing two genetic diseases in the same patient using NGS techniques is discussed.

Li, L., et al⁴⁶, Lal, D., et al⁴⁷, Cordova-Fletes, C., et al⁴⁸, Klimkowicz-Mrowiec, A., et al⁴⁹, Manzanilla-Romero, H.H., et al⁵⁰, Fabrizi, G.M., et al⁵¹, Melchionda, L., et al⁵², Maselli, R.A., et al⁵³, Delmiro, A., et al⁵⁴ and Coussa, R.G., et al⁵⁵ report cases in which NGS techniques allowed the identification of variants in more than one gene that can explain the patient’s phenotype.

These are the cases most similar to the described patient.

In Hata, Y., et al³⁹, NGS techniques allowed not only the concomitant diagnosis of Dravet syndrome and recessive symptomatic focal epilepsy with mental retardation in one of the reported patients, but also the identification of variants in genes associated with other diseases.

Even though the patients in the study died at an early age (before these other disorders could develop and become relevant), the identification of the correlation between Dravet syndrome and variants in cardiac disease-related genes may allow better treatment and follow-up of other patients with epilepsy in the future. Manzanilla-Romero, H.H., et al⁵⁰ gives another example of a patient in whom an increased risk for an unrelated disease was identified. This occurred because a mosaicism containing 2 genes was diagnosed in a tissue not directly affected by the disease.

Armour, C.M., et al⁵⁶ managed to breakdown a poligenic syndrome – Fitzsimmons syndrome - into the various affected genes and respective phenotypic repercussions. This syndrome encompassed the neurological symptoms of cognitive impairment, slowly progressive lower- limb spasticity and dysarthria, as well as skeletal abnormalities like brachydactyly with cone shaped epiphyses and short stature. However, it was demonstrated that the neurological symptoms were, in fact, due to variants in the SACS gene, while the skeletal abnormalities were associated with a variant in the TRPS1 gene that causes trichorhinophalangeal syndrome type 1 (TRPS type 1). TRPS type 1 is an autosomal dominant disorder⁵⁷ that leads to brachydactyly and other skeletal abnormalities that were also present in patients diagnosed with Fitzsimmons syndrome.

NGS techniques are also used to understand the genetic basis of multifactorial diseases, like autism-spectrum disorder (and its association with epilepsy and macrocephaly) and hippocampal sclerosis, as is shown in the articles Marchese, M., et al⁵⁸ and Azakli, H., et al⁵⁹.

17 Some diseases, like Bardet-Bield syndrome (BBS) are polygenic. The use of NGS techniques in those cases allows us to make more accurate genotype-phenotype correlations according to the affected genes. This can be seen in the articles Chakrabarty, S., et al⁶⁰ and Beigi, F., et al⁶¹. In the articles Melchionda, L., et al⁵² and Uittenbogaard, M., et al⁶², it is demonstrated that mutations in certain unrelated genes can influence the phenotype of genetic diseases. Tsurusaki, Y., et al⁶³ also reports a case where non pathogenic mutations influenced the patient’s phenotype, even though no diagnosis was achieved. The use of NGS techniques facilitates the identification of these situations.

18

D ISCUSSION

We describe a 50 years old female, born from consanguineous parents, with epilepsy during childhood and spastic-ataxia developing in early adulthood, followed by neuropathy, oculomotor apraxia and mild dystonic posturing. On MRI hyperintensities of the posterior arms of the internal capsule, tranversepathways of the pons, median brainstem raphe and middle cerebellar peduncules were present. Also progressive cerebellar atrophy was documented on the two exams. A sensorimotor neuropathy of predominant axonal type was documented on EMG. Albumin was on the lower range of normal.

On NGS panel for autosomal recessive ataxias, an homozygous variant in SACS c.4521_4522del(p.(Asn1508Serfs*32)) was identified, classified as likely pathogenic due to producing a truncated protein with a probable loss of function. It would have been relevant to study the patient’s parents to understand the segregation process. Nonetheless, this was not possible. This allowed us to perform a diagnosis of ARSACS. Her phenotype was compatible with ARSACS, having the classic triad of cerebellar ataxia, peripheral polyneuropathy and lower limb spasticity. Even though onset is typically in infancy, there are reports of patients whose symptoms only developed later, in the fourth-fifth decade of life. Epilepsy has been described in ARSACS but the large interval this patient had until motor symptoms onset made us question if it could be part of the phenotype. Nystagmus is part of ARSACS phenotype, and it’s also common for lower limb spasticity to disappear as peripheral polyneuropathy progresses. As for the MRI, two findings associated with ARSACS were present: hyperintensities in the middle cerebellar peduncles and cerebellar atrophy that was more pronounced in the superior vermis.

ARSACS peripheral neuropathy is most commonly of demyelinating nature and progresses to a mixed one. However, pure axonal neuropathies, like the one this patient has, have also been reported in ARSACS.

Two variants in compound heterozygosity were also identified in APTX gene. The first variant, c.837G>A(p(Trp279)), had already been described as pathogenic. The second, c.762G>A(p.(=)), predicted by bioinformatics tools to cause a change in splicing, was classified as VUS. Splice-site mutations have been described in AOA1 but without functional studies no further conclusions can be drawn with certainty. Measurement of APTX levels in fibroblasts would be of upmost importance but unfortunately could not be performed yet. Also, the study of other family members (for segregation analysis) would be important, but both her parents have passed away

19 and her half-siblings were not willing to collaborate. Still, we discuss it from a phenotypic point of view. Disease onset is not suggestive of AOA1, since its symptoms usually start during childhood. However, it is true that there have been some rare reports of very late AOA1 onset, which would be compatible with this patient. The patient presents cerebellar ataxia, oculomotor apraxia and peripheral polyneuropathy. Cerebellar ataxia and peripheral polyneuropathy may be identified in both AOA1 and ARSACS, while oculomotor apraxia is more specific of AOA1. This patient’s albumin levels were on the lower range of normal, but she had marked lower limb edema. There are no specific signs for AOA1 on brain MRI with most patient exhibiting cerebellar atrophy, a finding that is common to most cerebellar ataxias.

20

C ONCLUSION

With this work we would like to highlight the challenges posed by NGS in interpreting novel variants. The described patient had variants in SACS and APTX, two genes with partially overlapping phenotypes. For both she has an atypical, late onset, but only a diagnosis of ARSACS can be performed with certainty, due to the presence of a pathogenic variant in homozygosity.

This case illustrates one of the limitations of NGS techniques, as it can have results that physicians may have a hard time interpreting. However, it also shows one of its benefits, because one of these diagnoses would likely be missed if NGS techniques weren’t being used.

Just like in this case, there have been other case reports of NGS techniques being applied either for the same purpose, or for another. Whatever the context is, it’s important to understand that these studies, just like every other exam, have drawbacks that clinicians should be familiar with in order to reduce the drawbacks of using this powerful tool.

Finally, this is the first report of the likely pathogenic variant c.4521_4522del(p.(Asn1508Serfs*32)) in SACS.

21

T ^ABLES

Table I – Patients with neurological disorders and pathogenic variants in two or more genes

Legend: MELAS - Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes; HSP – hereditary spastic paraplegia; LGMD2H/STM – limb-girdle muscular dystrophy type 2H/

sarcotubular myopathy; ARSACS - Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay; TSC – Tuberous Sclerosis Complex; CMT2 - Charcot-Marie-Tooth disease type 2; CFTD – congenital fiber-type disproportion; ALS - Amyotrophic Lateral Sclerosis; BBS - Bardet-Biedl Syndrome; VUS – variants of unknown significance

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Myotonic dystrophy type 1

• Posterior cataracts

• Handgrip weakness

• Myotonia

• Scapular winging

• Full/ Everted lips

• 800 CTG repeats in the DMPK gene

• A deleted fragment in the D4Z4 macrosatellite

A concomitant diagnosis of myotonic dystrophy type 1 and fascioscapulohumeral muscular dystrophy type 1 was made

To find a second diagnosis

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

MELAS • Worsening muscle fatigability and weakness

• Atrophy of the right pectoralis

• High riding scapula with arm abduction

• Asymmetric weakness and atrophy of the periscapular muscles

• m.3243A>G

• A deleted fragment in the D4Z4 macrosatellite

A concomitant diagnosis of

MELAS and

fascioscapulohumeral muscular dystrophy type 1 was made

To find a second diagnosis

22 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Becker muscular dystrophy

• Mild bilateral calf pseudohypertrophy

• Premature birth (32 weeks) with macrosomia

• Pregnancy complicated by polyhydramnios

• Undescended testis and umbilical hernia during childhood

• Obesity

• Pit on the right ear

• Crease on the left ear

• Macroglossia

• Hemizygous in-frame deletion of exons 45-48 of the DMD gene

• Hypometilation at IC2 of

the maternal

chromossome

A concomitant diagnosis of Becker muscular dystrophy and Beckwith-Wiedemann syndrome was made

To find a second diagnosis

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Duchenne muscular dystrophy

• Mild proximal weakness

• Bilateral calf pseudohypertrophy

• Single-touch Gower’s maneuver

• Mild waddling gait

• Stiffness since birth

• Falls without defensive posturing

• An exaggerated startle response

• Awakening with significant seizure- like shaking

• Positive nose tapping test

• DMD deletion of exons 8-47

• Pathogenic variant in exon 8 of the GLRA1 gene (c.920A > G, p.

Tyr307Cys)

A concomitant diagnosis of Duchenne muscular

dystrophy and

hyperekplexia was made

To find a second diagnosis

23 Table 1 (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Myotonic dystrophy type 1

• Hypotonia

• Intellectual disability disproportionate to the diagnosis of myotonic dystrophy type 1

• 800 CTG repeats in the DMPK gene

• Partial deletion of NRXN1 gene

A concomitant diagnosis of myotonic dystrophy type 1 and microdeletion syndrome involving NRXN1 was made

To find a second diagnosis

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Myotonic dystrophy type 1

• Global development delay

• History of seizures

• Low vision

• Congenital heart defects (coarctation of the aorta and atrial and ventricular sept defects)

• Multiple dysmorphic facial features

• Short stature

• Clinodactyly

• Brachydactyly

• <500 CTG repeats in the DMPK gene

• Two de novo microdeletions on chromosome 1

A concomitant diagnosis of mild myotonic dystrophy type 1 and microdeletion syndrome was made

To find a second diagnosis

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Duchenne muscular dystrophy

• Modified Gower maneuver

• Global development delay preceding the onset of typical features of Duchenne muscular dystrophy

• Wheelchair use at the age of 8

• Pathogenic variant in exon 70 of the DMD gene

• Duplication at 1q21.1q21.2

A concomitant diagnosis of Duchenne muscular dystrophy and 1q21.1 microduplication syndrome was made

To find a second diagnosis

24 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Hannah- Shmouni, F., et al³⁸ [2021]

(8 patients)

Spinocerebellar ataxia type 14

• Development delay

• Intellectual disability

• Spasticity

• Upgoing toes

• Ataxia

• Dysmorphic facial features

• Pathogenic variant in the PRKGC gene

• Partial deletion of the SOX5 gene

A concomitant diagnosis of spinocerebellar ataxia type 14 and Lamb-Shaffer syndrome was made

To find a second

diagnosis

Hata, Y., et al³⁹ [2020], (2 patients)

Dravet syndrome • Mild development delay

• Frequent seizures

• Novel variant in intron 23 of SCN1A gene

• Heterozygous for two variants in the CNTNAP2 gene

• MYBPC3_T1046M

• CETP_c.1146 + 1G > A

• TCAP_E49K

• Concomitant diagnosis of Dravet syndrome

and recessive

symptomatic focal epilepsy with mental retardation was made

• Identification of variants associated with increased risk of hypertrophic

cardiomyopathy, dilated cardiomyopathy and cholesterol ester transfer protein deficiency (the patient died before the age of disease onset)

• To find a second diagnosis

• To find variants associated with other diseases

25 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Hata, Y., et al³⁹ [2020], (2 patients)

Dravet syndrome Frequent febrile seizures • Missense variant in SCN1A

• ZMPSTE24_L438F

• SYNE2_T5138M

Increased risk of metabolic disease and dilated cardiomyopathy and Emery-Dreifuss muscular dystrophy-5 (the patient died before the age of disease onset)

To find variants associated with other diseases

Wayhelova, M., et al⁴⁰ [2020] (2 patients)

Biotinidase deficiency

• Intelectual disability with autistic features

• Microcephaly

• Cortical blindness

• Central hypotonia

• Refractory atonic seizures

• Hemizygous for a deletion in exon 5 of the IQSEC2 gene

• Heterozygous

pathogenic variant in the BTD gene

A concomitant diagnosis of biotinidase deficiency and non-syndromic X-linked intellectual development disorder-1

To find a second diagnosis

Wayhelova, M., et al⁴⁰ [2020] (2 patients)

Biotinidase deficiency

• Intelectual disability

• Central hypotonia

• Strabismus

• Refractory atonic and myoclonic seizures

• Hemizygous for a deletion in exon 5 of the IQSEC2 gene

• Heterozygous

pathogenic variant in the BTD gene

A concomitant diagnosis of biotinidase deficiency and non-syndromic X-linked intellectual development disorder-1

To find a second diagnosis

Wiedemann, A., et al⁴¹ [2020] (2 patients)

HSP • Spastic paraplegia

• Hyperreflexia

• Bilateral extensor plantar responses

• Cognitive impairment

• Compound heterozygous variants in MTHFR

• Two rare pathogenic variants in POLG

A concomitant diagnosis of MTHFR deficiency and a POLG-related syndrome was made

To find a second diagnosis

26 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Wiedemann, A., et al⁴¹ [2020] (2 patients)

HSP • Spastic paraplegia

• Hyperreflexia

• Bilateral extensor plantar responses

• Cognitive impairment

• Compound

heterozygous variants in MTHFR

• Two rare pathogenic variants in POLG

A concomitant diagnosis of MTHFR deficiency and a POLG-related syndrome was made

To find a second

diagnosis

Marttila, M., et al⁴² [2019]

(1 patient)

Congenital myopathy

• Generalized hypotonia

• Diminished reflexes

• Muscle weakness

• Homozygous for a pathogenic variant in MYL2

• Heterozygous for a pathogenic variant in NEB

• Heterozygous for a benign variant in the TTN gene

• Hemizygous for a likely benign variant in the DMD gene

A diagnosis of CFTD and

infantile onset

cardiomyopathy associated with pathogenic variants in the MYL2 gene was made

To find a second

diagnosis

27 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Dangiolo, S.B., et al⁴³ [2015] (1 patient)

Clinically diagnosed Bohring- Opitz syndrome

• Neurodevelopment delay

• Failure to thrive

• Pharyngeal dysphagia

• Gastro-esophageal reflux

• Severe scoliosis

• Abnormal uterine bleeding

• Severe recurrent acute pancreatitis

• Microcephaly

• Dysmorphic facial features

• Hypermobile joints

• Flexion contractions in the distal parts of all limbs

• Tremor

• Choreiform movements

• A frameshift variant in the ASXL1 gene

• Compound

heterozygosity in the CFTR gene

A concomitant diagnosis of Bohring-Opitz syndrome and cystic fibrosis was made

To find a second diagnosis

Moteki, H., et al⁴⁴

[2015] (2

patients)

MELAS • Progressive bilateral

neurosensorial hearing loss

• Diabetes mellitus

• Hypertrophic cardiomyopathy

• Overweight

• Short stature

• mtDNA 3243A>G variant

• Missense variant in the P2RX2 gene

A concomitant diagnosis of MELAS and autosomal dominant deafness-41 was made

To find a second diagnosis

28 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Moteki, H., et al⁴⁴ [2015] (2 patients)

MELAS • Progressive bilateral

neurosensorial hearing loss

• Diabetes mellitus

• Hypertrophic cardiomyopathy

• Mental retardation

• Overweight

• Short stature

• mtDNA 3243A>G variant

• Missense variant in the P2RX2 gene

A concomitant diagnosis of MELAS and autosomal dominant deafness-41 was made

To find a second

diagnosis

Nectoux, J., et al⁴⁵ [2015] (2 patients)

Non identified limb-girdle

muscular dystrophy

• Pelvic and lower-limb muscular weakness associated with atrophy

• Lower-limb arreflexia

• Distal upper-limb atrophy

• Cognitive impairment

• Homozygous deletion of the whole TRIM32 gene

• Homozygous partial deletion of the ASTN2 gene

A concomitant diagnosis of

LGMD2H/STM and

cognitive impairment related to ASTN2 was made

To find a second

diagnosis

Nectoux, J., et al⁴⁵ [2015] (2 patients)

Non identified limb-girdle

muscular dystrophy

• Progressive muscle weakness with frequent falls

• Scapular winging

• Waddling gait

• Heterozygous

deletion of the whole TRIM32 gene

• Heterozygous partial deletion of the ASTN2 gene

A diagnosis of

LGMD2H/STM was made

To find a second

diagnosis

Li, L., et al⁴⁶ [2019] (1 patient)

Phenylketonuria • Generalized hypotonia

• Hearing loss

• External ophthalmoplegia

• Pronated feet

• Mental retardation

• Delayed development

• Compound

heterozygous in the PAH gene

• Compound

heterozygous in the POLG gene

The patient’s phenotype was explained by concomitant

phenylketonuria and POLG- related syndrome

To find

variants in multiple genes that explain the phenotype

29 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Lal, D., et al⁴⁷ [2016] (2 patients)

Hereditary

hypophosphatemic rickets

• Muscular weakness

• Delayed motor milestones

• Muscular hypotonia

• Rigid spine

• Short stature

• X-legs

• Respiratory failure

• Homozygous variants in SEPN1

• Homozygous variants in SLC34A3

The patient’s phenotype was explained by concomitant recessive SEPN1 and SLC34A3 disease

To find

variants in multiple genes that explain the phenotype

Lal, D., et al⁴⁷ [2016] (2 patients)

Hereditary

hypophosphatemic rickets

• Muscular weakness

• Muscular hypotonia

• X-legs

• Homozygous variants in SEPN1

• Homozygous variants in SLC34A3

The patient’s phenotype was explained by concomitant recessive SEPN1 and SLC34A3 disease

To find

variants in multiple genes that explain the phenotype

30 Table I (continued)

Article and nº of patients

Disease Symptoms/ Signs Genetic variants Diagnostic changes post-

NGS

Benefit of NGS

Cordova- Fletes, C., et al⁴⁸ [2018] (1 patient)

Brain anomalies and skeletal dysplasia of unknown origin

• Mental retardation

• Short stature

• Microcephaly

• Pulmonary valve stenosis

• Kyphoscoliosis

• Seizures

• Chronic constipation

• Generalized hypertonia

• Generalized hyperreflexia

• Multiple dysmorphic facial features

• Micropenis

• Phimosis

• Cryptorchidism

• Shawl scrotum

• Short hands

• Brachysyndactyly

• Symphalangism

• Homozygous likely pathogenic variant in the SPAG17 gene

• Homozygous VUS in the WDR35 gene

• VUS microduplication of the NXPH3 gene

The patient’s phenotype was explained by concomitant SPAG17 and WDR35 related disease

To find variants in multiple genes that explain the phenotype

Klimkowicz- Mrowiec, A., et al⁴⁹ [2021]

(2 patients)

Frontal variant of Alzheimer’s disease

• Apathy

• Episodic memory impairment

• Executive dysfunction

• Rare polymorphism in the PSEN2 gene

• Pathogenic variant in the GRN gene

The diagnosis was changed from frontal variant of Alzheimer’s disease to frontotemporal dementia

To find variants in multiple genes that explain the phenotype