Identification of Pathogenic variants in IGHMBP2 gene and Application on Prenatal diagnosis of Rare Neuromuscular Disorders

of Rare Neuromuscular DisordersABSTRACT Background spinal muscular atrophy with respiratory distress type 1 SMARD1 and Charcot - Marie - Tooth type 2S CMT2S are rare neuromuscular disord

IDENTIFICATION OF PATHOGENIC VARIAN TS

IN IGHMBP2 GENE AND APPLICATION ON

PRENATAL DLAGNOSIS OF RARE NEUROMUSCULAR DISORDERS

Graduation ThesisMajor: General PractitionerMajor code: 52720101

Academic Supervisor

Associate Professor TRAN VAN KHANH M.D, PhD

ACKNOWLEDGEMENT

Firstly I would like to express my deepest gratitude to Associate Professor TranVan Khanh MD PhD Deputy Director of Center for Gene-Protein Research Head ofMolecular Pathology Department of Hanoi Medical University, who provided me greatsupport and leadership which allowed me to conduct the research and finish this diesissuccessfully I am thankful to her for presenting such excellent advice and guidancedespite having a tight schedule

I heartily thank Professor Ta Thanh Van, M.D, Ph D Chairman of UniversityCouncil of Hanoi Medical University and Director of die Center for Gen-ProteinResearch Professor The-Hung Bui MD, PhD Centre for Molecular Medicine ClinicalGenetics Unit in Karolinska Universitat, and Assc Professor Tran Huy Thinh M.D., Ph

D Deput)' Head of Department of Biochemistry Head of Department of Technologyand Scientific Research Management, who always created favorable conditions for me

to access scientific research and inspired me to be a good doctor and researcher with athorough understanding of medical ethics

I am deeply thankful to Dr Luong Hoang Long Department of Clinical Allergy Immunology and Dermatology National E hospital for his patience, consistent motivation and

immense knowledge He also always provided me with

insightful comments and hard questions during my

scientific research projects

-ÍM Qỉ ugc V Hl

molecular techniques.

The last words of thanks 1 would like to send to my parents, who are the idols

in me and have always laid concrete encouragement to me in my life

Hanoi May 2021

Cao Ha My

I hereby certify that this thesis incorporates original research, which has not been previously submitted for a degree to any other institution, and the best of my

knowledge and belief, it does not contain any material previously published or written

by other persons except where reference has been made in the text

Hanoi May 2021

CAO HA MY

IVF in vứro Fertilization

RNA Ribonucleic Acid

INTRODUCTION

Table 1.1 List of genes identified as distal hereditary motor neuropathies (dHMN)

of Rare Neuromuscular Disorders

ABSTRACT

Background spinal muscular atrophy with respiratory distress type 1 (SMARD1) and Charcot - Marie - Tooth type 2S (CMT2S) are rare neuromuscular disorderscaused by biallelic pathogenic variants on IGHMBP2

Objectives: 1) To identify pathogenic variants in IGHMBP2 gene and describe four cases with IGHMBP2 mutation 2) To identify’ earners and application on prenatal

diagnosis

Subjects and methods: Four patients under 12 years of age with lower limbsweakness (3 4 had respiratory disorders) and family members Genetics analysis forpatients and family members was performed using Next Generation Sequencing andSanger Sequencing

Results We identified four IGHXỈBP2 mutations, in which C.1574T> c (p

Leu525Pro) is a novel mutation Both parents and sisters of four patients were identified

to be carriers The mother of the third patient’s family was pregnant but had an abortionafter genetics testing of the fetus revealed compound heterozygous mutations on

IGHMBP2.

Conclusion: 1 patient was homozygous for IGH.\iBP2 mutation, and three out of

4 patients had compound heterozygous mutations All parents were identified ascarriers, and we successfully applied the genetic testings on prenatal diagnosis for thefamily of patient 03

respiratory distress Charcot-Marie-Tooth SMARD1

INTRODUCTION

Neuromuscular disorders (NMDs) include various conditions that affectcomponents of a motor unit, sensor.' and autonomic nen es, or their supportive

r-u -ÍM CỊỈ ugc V Hl

r-u -ÍM CỊỈ ugc V Hl

condition, trauma, nutritional deficiency to the hereditary cause The presence of NMDincurred health burden not only for the child’s disability and early death, lower quality

of life but also a financial burden for the family and society, and most importantlyemotional burden and stigma for the parents

Congenital NMD, or hereditary NMD is relatively rare Therefore the chance ofencountering a case in daily clinical practice is meager Physicians would hardlyencounter such a case, and when they do the condition is often misdiagnosed,especially in Vietnam where many neuromuscular disorders were misdiagnosed aseither cerebral palsy or myasthenia gravis Definitive diagnosis is crucial for providingthe correct treatment and counseling direction for the patient In addition research at themolecular level will unravel the mechanism of the disease, enabling scientists toelaborate effective therapy for patients in the future

/GẢBÍ5P2-related NMD is a congenital disease which was reported in twoclinical manifestations: SMARD1 and CMT2S with relatively different phenotype andprognosis Differentiated and definitive diagnosis of these two diseases required bothextensive clinical examination and identification of genetic defects Deciphering theexact pathogenic variants allows for thorough genetic counseling, prenatal diagnosisand pre-implantation genetic diagnosis for high-risk families Since there haven’t beenany curative treatments available for any NMD, detection of disease-causing variantsallowed physician to provide medical support (such as prenatal diagiosis) to family withhope of having a healthy child

In order to better understand the natural history and mechanism of

ĨGHMBP2-related NMD and other rare diseases, it is necessary to create a domestic rare diseasenetwork with a large cohort and longitudinal follow-up Currently, some internationaland regional rare disease network has already been established with the aim for helpingdoctors and patients to raise awareness about the rare disease Therefore, our research on

IGHMBP2-related NMD will contribute initially to building the database of rare

diseases in Vietnam and a premise for further studies in the field of rare diseases,

Application on Prenatal Diagnosis of Rare Neuromuscular Disorders" was conductedwith two primary objectives.

manifestations of the patients

To detect carriers among family members and apply on

prenatal diagnosis

r-u -ÍM CỊỈ ugc V Hl

r-u -ÍM CỊỈ ugc V Hl

C HAPTER 1 LITERATURE REVIEW

The primary function of the peripheral nervous system (PNS) is to transferinformation from the limbs to and from the central nervous system (CNS), whichconsists of the brain and the spinal cord The nen es responsible for this include thecranial nen es that link to the brainstem and 31 pairs of spinal nerves that branch outbetween each vertebra of the spine, connecting to the spinal cord Components of thePNS are the sensory nen es, the motor nen es, and the autonomic nenous system Thesensory nen es are the afferent nen es that convey information from the sensory organsand limbs to the brainstem and the spinal cord, which typically have cell bodies located

in the dorsal root ganglia located close to the spinal cord Meanwhile, the motor nenesarising from the ventral horn of the spinal cord transfer information from the brainstemand the spinal cord to the neuromuscular junctions at the muscles The autonomicnervous system is also considered part of the PNS as it works in conjunction with thesensory and motor nen es However, the peripheral nenes include not only the nene fibersbut also several layers of connective tissue (endoneurium perineurium, and epineurium)and blood vessels [1] [2]

Individual nene fibers consist of long axons extending to the body's extremitiesthat may be myelinated or unmyelinated Myelin in the peripheral nervous systemderives from Schwann cells, which adhere to nene cell membranes and create multiplelayers or wrappings of the membrane This myelin sheath results in an insulating lipid-rich layer around the nene fibers, allowing for a higher conduction velocity depending

on the diameter of the sheath Unmyelinated axons are solely enveloped by a singlelayer of Schwann cell cytoplasm Thus they conduct very slowly by a continuous mode

of propagation of the electrical signal In the context of peripheral neuropadlies,abnormalities can be found in both the axon and the myelin sheath, causing differentphenotypes [1]

The Motor unit is physiologic Typically, striated muscle contraction is only

possible through the firing of motor neurons that are activated either via descendingpathways or through reflex connections The muscle fibers in a motor unit respond in anall-or-none fashion to excitation by the motor neuron, producing a quick twitch.Conditions that damage some motor units (sparing others) usually result in overallweakness of the muscle but high firing rates of individual motor units that are still intactThis is because of the decreased number of motor units that must be activated atmaximal frequencies to generate any muscle force Therefore, weakness with a highfiring rate indicates a loss of motor neurons or motor axons [2]

Neuromuscular disorders

There has not been a unanimous classification of Neuromuscular disordersHowever based on anatomical characteristics of the PNS, there are three basic types ofneuromuscular disorders, including damage to anterior horn cells (also called motorneuron disease), damage to the peripheral nene fibers (myelin or axons), and damage tothe neuromuscular junction The etiology of this disease could either be congenital orsecondary' to prior medical conditions or acquired during a lifetime Thus, the termcongenital (or inherited) muscular disorders share some similarities with neuromusculardisorders and include other types of diseases Currently, the congenital muscular disordercan be divided into congenital neuromuscular disorder and congenital myopathyMyopathy comprises two types: the disease that progressively destroys muscle fiberssuch as Duchenne muscular dystrophy, Limb-Girdle muscular dystrophy and diseaseleading to congenital functional defect of the muscle without distinct muscle wasting areusually disease of the ion channel The following paragraphs will introduce the basicclassification of neuromuscular disorders

1.1.2.1 Motor neuron disease

Motor neuron disease results from damage to the anterior horn cells arising fromthe spinal cord to the limbs (lower motor neurons) or the upper motor neurons of thecerebral cortex that give rise to the descending tracts that control movements Theseconditions can present weakness accompanied by lower motor sign s (atrophy,fasciculations and decreased reflexes) or upper motor neuron signs (spasticity, increased

r-u -ÍM Qỉ ugc V Hl

r-u -ÍM CỊỈ ugc V Hl

reflexes, and upgoing toes) The most common condition affecting both the upper andlower motor neurons is amyotrophic lateral sclerosis ALS There are also variants ofmotor neuron disease, which selectively involve the upper motor neurons (primarylateral sclerosis), the lower motor neurons (progressive muscular atrophy), or the cranialmusculature (bulbar palsy) The cause for motor neuron disease can be genetic defects,termed Hereditary Motor Neuropathy (HMN) For this thesis, a specific type of HMN(called Spinal Muscular Atrophy with Respiratory distress) will be further discussed inthe following sections.

1.1.2.2 Peripheral neuropathy

Peripheral neuropathy is a disease that affects the peripheral nerve fibers, whichincludes mononeuropathy, mononeuropathy multiplex, or polyneuropathy- Therefore.both abnormal motor and sensory features can be presented The symptoms candetermine the particular nerve or nerves that are affected Symptoms may be "positive"(including pain and dysesthesia), may be harmful (including loss of sensation, weakness,

or loss of reflexes), or may be irritative (such as fasciculationsor paresthesias)

Mononeuropathy and radiculopathy are most often due to ưauma of some types The most common mononeuropathies are the elbow and radial nene damage fracture of the humerus Radiculopathy indicates damage to nene root(s) and

typically occurs as a component of several spinal diseases

In younger individuals, this is usually due to

intervertebral disc herniation This is more often due to degenerative changes in the discs, bones and joints in older individuals 'Mononeuritis multiplex" is a relatively rare presentation of certain disorders that damage nerves primarily by interfering with blood flow to nerves or plexi

or by an autoimmune process damaging either the myelin or axon The most common cause is diabetes mellitus

Polyneuropathy can affect either the axon, or myelin sheaths (demyelinating), orboth There are over 100 known acquired and inherited disorders that may causepolyneuropathy Since the nen es to the lower limbs are the longest they are the mostdependent on a good supply of metabolic substrates and have the most significantexposure to toxins or damaging the myelin Therefore, symptoms and signs are mostprominent in the feet Loss of sensation ("numbness”) is the most common finding butparesthesias or dysesthesias (prickling, tingling, burning, etc.) are also common Causesfor polyneuropathy can be categorized as metabolic, nutritional (deficiency of vitaminB) toxic (alcohol), infection (HIX’ syphilis), inflammatory, dysproteinemia andhereditary There is an incredible number of peripheral neuropathies that may be familial: some of the most common is Charcot Marie-Tooth disease with around 24 subtypes[1], [3] [4], For this thesis Charcot- Marie-Tooth will be further discussed in thefollowing parts

LI.2.3 End-plate (neuromuscular junction)

In most cases, each muscle fiber has only one end-plate, which plays as the plug

for the nerve terminal branch Acetylcholine is the neurotransmitter produced from themotor neuron stimulating specific muscles Myasthenia gravis is the most commondisorder affecting neuromuscular transmission Autoantibodies primarily mediate thedisease against die Acetylcholine receptor (AchR) The impairment of neuromusculartransmission and muscular weakness is explained by several mechanisms includingfunctional blockade of AChR increased degradation of AChR and die complement-mediated destruction of the postsynaptic folds [5], 1.1.3 Spinal Muscular Atrophy with

Respiratory distress 1 (S.MARD1) LỈ.3.Ỉ Overview of distal hereditary- motor

neuronopathy

The distal hereditary motor neuronopathies (dHMN) are a geneticallyheterogeneous group of diseases characterized by distal lower-motor-neuron weaknessdHMN is also refened to as distal spinal muscular atrophy (dSMA) a reflection of thecommonly held but unproven belief that the pathology resides in the ventral horn of thespinal cord of note dHMN is often referred to as a 'neuronopathy' instead of a'neuropath/ based on the hypothesis that the primary pathologic process resides in theneuron cell body and not in the axons [6]

The dHMN usually presents as a classical peroneal muscular atrophy syndromewithout sensory symptoms [4] The overall clinical picture consists of progressiveweakness and wasting of the extensor muscles of the toes and feet Later on weaknessand wasting also involve the distal upper limb muscles Foot deformity is a commonfeature Often, unusual or additional features are present in ‘complicated' distal HMN.including predominance in the hands, vocal cord paralysis, diaphragm paralysis, andpyramidal tract signs In some families, several of these additional signs co-occur

In one of the earliest reports on distal HMN, a family was described with an autosomal dominant mode of inheritance and onset in early adult life Also, a kinship with

autosomal recessive inheritance and isolated cases were reported (7) By then, affected individuals with variable onset ages, ranging from infancy to adult life, were

documented The diagnosis of dHMN in a patient with a distalmotor neuropathy phenotype first requires considering

causing variant is not always straightforward owing to de novo mutations, small families, and non-paternity A

detailed history, as is often the case, is most

informative The cardinal feature is usually a slowly

progressive lengthdependent condition, often starting in the first two decades while the third decade is not

uncommon Poor performance in school and insidious

progression are valuable clues, whereas a short, de novo history in middle age should prompt a search for an

acquired etiology Bulbar involvement other than the

recurrent laryngeal nerve, is rare in dHMN The examination usually confirms distal wasting and weakness with reduced

or absent reflexes, and neurophysiology confirms reduced motor amplitude potentials associated with EMG changes suggesting chronic distal predominant denervation A

significant proportion of patients classified as dHMN will

be ‘sporadic’ with no apparent family history' using this approach

Theoretically dHMN is in contrast to Charcot-Marie-Tooth disease (CMT) and thehereditary sensory neuropathies where sensory involvement forms a significantcomponent of the disease [4] Nevertheless, on clinical examination dHMN can hardly

be distinguished from Charcot-Marie-Tooth (CMT) neuropathy (hereditary motor andsensory neuropathies HMSN) because unmistakable sensory- signs are often lacking inthese disorders Many forms of dHMN have minor sensory' abnormalities, and there is an

r-u -ÍM Qỉ ugc V Hl

r-u -ÍM CỊỈ ugc V Hl

overlap between the axonal CMT (CMT2) and dHMN where the pathogenic variants in

the same gene (IGH.\!BP2) may cause both phenotypes (8] [9] Also, it is not uncommon

for patients with CMT2 and significant sensory involvement neurophysiologically tohave no sensory symptoms and minimal sensory signs Therefore, nene conductionstudies (NCS) are essential to support the diagnoses of CMT or distal HMN [10] Figure1.1 illustrated a Venn diagram of disease genes for CMT1 CMT2 and dHMN Thegenetic defect of dHMN and CMT will be further elaborated in the following sections.dHMN should also be differentiated with distal myopathy using electromyography(EMG) Like dHMN the distal myopathies are a genetically and phenotypically diversegroup of conditions Some such as Myoshi myopathy, may have neck flexion weaknesshelpful in making the diagnosis, but others may present with isolated foot drop In suchscenarios EMG is the most helpful test In the upper limbs, the intrinsic hand musclesare usually affected first in dHMN whereas in the distal myopathies, it is often theforearm flexors

1.1.3.2 Classification of dHMX based on genetic etiology

Based on age at onset, mode of inheritance, and additional [4], [12] However, some distal HMN families did not fit into the existing classification and represented novel clinical and genetic entities The genetic etiology of dHMN subtypes' genetic etiology- seems to be as

heterogeneous as their clinical manifestations Table 1.1 was reproduced from Rossor et al (2012) and Rudnik-

Schonebom et al (2020) which updated currently known genesrelated to this condition (11], [12]

Axonal CMT

Demyelinating CMT

u> wti rexoM mri

UCS»T OCỈHI artiOHt.

UCOJ WMtt UK MWH n.*rrĩẠ

Hereditary motor neuropathy

Figure 1.1 Venn diagram of disease genes for Charcot-Marie-Tooth disease

(subdivided into demyelinating and axonal CMT) and distal hereditary motor

neuropathy (dHMX) .AD = autosomal dominant; AR = autosomal recessive, XL = X-linked (reproduced from Sabine Rudnik-Schoneborn et aL, 2020) Ị1ỈỊ

r-u -ÍM Qỉ ugc V Hl

r-u -ÍM CỊỈ ugc V Hl

Table J.1 List of genes identified as distal hereditary motor neuropathies

(dHM.X) based on the original classification by Harding and Thomas 1980

(Reproduced from Rosso et al., 2012) [12]

HSPB1

weakness

HSPBS GARS DÌXCQHQ HSPBÌ

weakness

HSPBS BSCL2 HSPB3

Slowly progressive wasting and weakness

unknown Slowly progressive wasting and

GARS BSCL2

Spinal muscular atrophy with

DC7N1 TRPV4

dHMN and

SETX BSCL2

Congenital distal

spinal muscular

atrophy

1.1.3.1 Spinal Muscular Atrophy with Respiratory dìsưess ỉ (SMARD1)

SMARD 1 (OMIM 604320) also known as distal spinal muscular atrophy type 1

(DSMAl) or hereditary motor neuroncpathy type VI (dHMN VI), caused by the defect of

iht IGHMBP 2 gene Biallelic mutations in this gene result in degeneration of a-motor

neurons in the brain stem and the anterior horns of the spinal cord, causing spinalmuscular atrophy with respiratory distress type In contrast to the classical s MN 1-dependent spinal muscular atrophy, the first and predominant symptom of SMARD1 isrespirators’ distress due to diaphragmatic palsy, which typically arises as early as in thefirst year of life Respiratory failure usually precedes weakness of the distal muscles,which manifests as hand drops, fatty pads, finger contractures, and talipes Muscularatrophy and weakness become generalized within months and can lead to completetetraplegia

Mellins et al (1974) and Bertini et al (1989) delineated diaphragmatic spinalmuscular atrophy (SMA) as a variant of infantile SMA (SMA1; 253300) [13], [14], Themost prominent symptoms are severe respiratory distress resulting from diaphragmaticparalysis with eventration shown on chest x-ray and predominant involvement of theupper limbs and distal muscles In contrast to classic SMA1 in diaphragmatic SMA theupper spinal cord is more severely affected than the lower section In a series of morethan 200 patients with early-onset SMA Rudnik- Schonebom et al (1996) found thatapproximately 1% presented with diaphragmatic SMA and did not have a deletion of thesurvival of motor neuron gene on chromosome 5q (11)

Gro-hmann et al (1999) reported on nine patients from 3 families with diaphragmatic SMA following autosomal

recessive inheritance They referred to this disorder as SMARD (spinal muscular atrophy with respiratory distress) (15) The three families were of Lebanese, German, and Italian origin, respectively In family 1 the parents werefirst cousins The first affected son died al 10 weeks of suspected sudden infant death syndrome (SIDS: 272120) One daughter presented, at the age of 6 weeks, with feeding difficulties and progressive respiratory distress

-■c -ÍM Qỉ ugc V Hl

-■c -ÍM Qỉ ugc V Hl

Chest x-ray showed the eventration of the diaphragn She developed progressivemuscular atrophy with complete paralysis of the upper and lower limbs and mildcontractures of die knee and ankle joints Three other sibs died of respirator}- failure at

an earl}- age Autopsy specimens showed neurogenic atrophy of skeletal muscle withoutsigns of reinnervation The diameter of the anterior of the spinal roots was reduced in theupper spinal cord

The remaining motor neurons showed chromatolysis In family 2 the first affectedchild had severe muscular hypotonia and died at 9 weeks of cardiorespirator}- failure Athird child had been mechanically ventilated since die age of 3 months Family 3 hadbeen reported in detail by Novelli et al (1995) Grohmann et al (2003) reported the

clinical features of 29 infants with SMARD1 confirmed by a mutation in the IGHMBP2

gene [9], [16] Intrauterine growth retardation, prematurity, weak cry and footdeformities were the earliest symptoms Most patients' clinical manifestation appearcdatthe age of 1 to 6 months with severe respiratory distress due to diaphragmatic paralysis,and progressive muscular weakness with predominantly distal lower limb muscleinvolvement Sensory and autonomic nen es were also affected in some patients, asdemonstrated by decreased pain perception, excessive sweating, constipation, andbladder incontinence

Grohmann et al (2001) demonstrated that SMARD type 1 results from mutations

in the gene encoding immunoglobulin mu-binding protein 2 ỰGHMBP2\ OMIM

600502) In 6 SMARD1 families Grohmann et al (2001) detected three recessivemissense mutations, two nonsense mutations, one frameshift deletion, and one splice

donor site mutation Mutations in mouse IGHMBP2 have been shown to be responsible

for spinal muscular atrophy in the ’neuromuscular degeneration' (nmd) mouse, whosephenotype resembles the SMARD1 phenotype Among 29 infants with SMARD1,

Grohmann et al (2003) identified 26 novel mutations in the IGHMBP2 gene, including

14 missense, six nonsense, four frameshifls one inframe deletion, and one frameshiftinsertion This gene is also associated with Charcot-Marie-Tooth type 2S (CTM2S),which leads to weakness of distal limbs due to axonal damage A detailed comparison ofthe clinical manifestations of CMT2S and SMARD1 can be looked up in Table 1 3 [9]Pitt et al (2003) reported 13 intanis with early-onset diaphragmatic palsy in

association with a progressive axonal neuropathy who showed similar characteristics.The authors stated that none of the patients shared the exact characteristics of patientswith SMARD1 most notably the absence of pathologic changes in anterior horn cells of

1 patient examined Pitt et al (2003) developed a set of diagnostic criteria to classify thesyndrome, including low birth weight (below the third percentile), onset within the firstthree months of life, early onset of respirator}’ compromise with ventilator dependence,and inability to wean slow’ motor nerve conduction velocities, and a general decrease inthe size of myelinated fibers on sural nene biopsy All eight patients tested were found to

have mutations in the IGHMBP2 gene, indicating that a broader spectrum of phenotypic

features may be associated with mutations in that gene (17]

To clinically delineate the various early- and late-onset forms of distal spinalmuscular atrophy from SMARD1 Guenther 2007 conducted a cluster analysis of

clinical symptoms showing that the probability of finding mutations in JGHMBP2 in

patients with respiratory' distress and suspected SMARD1 could be predicted by thefollowing items [18]:

(1) manifestation age of respiratory distress between 6 weeks and six months

(2) presence of diaphragmatic paralysis

(3) distal muscular weakness

(4) intrauterine growth retardation

1.1.4 Charcot-Marie-Tooth disease, type 2S

1.1.4.1 Overview of Charcoi-Marie-Tooilt disease

Charcot-Marie-Tooth (CMT), also known as Hereditary Motor SensoryNeuropathy (HMSN) functions as a term covering a group of clinically and geneticallyheterogeneous inherited neuropathies [19], The prevalence of CMT in the generalpopulation can vary but has an overall 1 in 6000 [20] Following the anatomical-basedclassification of Neuromuscular disorders, CMT belonged to the group of peripheralneuropathy Theoretically, it should be differentiated from distal hereditary motorneuronopathy (dHMN), a type of motor neuron disease with degeneration of the anteriorhorn of the spinal cord However, clinically, there has not been a clear distinctionbetween these two groups, for example, patients with dHMN on the first examination

-c -ÍM Qỉ ugc V Hl

-■c -ÍM CỊỈ ugc V Hl

can develop sensory symptoms later in life and be classified as CMT Some neurologistseven classify dHMN as the third type of CMT also known as spinal CMT besides thetwo major types CMT1 and CMT2.

Due to the phenotypic variability the classification of CMT is not only based onclinical presentation but also on neurophysiology’ or genetic testing CMT can bedivided into demyelinating CMT (CMT1) with degeneration of the myelin sheatharound the axon and axonal CMT (CMT2) with the loss of myelinated axons slightsegmental degeneration Generally, axonal loss occurs at the distal ends of fibers, aprocess called “axonal dying back." Motor nerve conduction velocity (MNCV) is a vitalelectrophysiology testing for the classification of CMT Uniformly slow MNCV Lessthan 38 ms in the arms is characteristic for demyelinating CMT1, while MNCV abovethis cut-oô' is typical of axonal CMT2 The intermediate form of CMT has intermediateelectrophysiological features, i.e., MNCV from 25 to 45 m s [21],

Besides the phenotypic variability seen in patients with CMT, a heterogeneous genotypic presence characterizes this disease Pathogenic variants inmore than 80 genes have been found so far and more are being unraveled The geneticbackground plays an essential role in classifying the disease and will be crucial to findcommon pathways to explain the characteristic features seen in most patients So far,there have been more than 20 types of CMT based on genetic features and nen econduction velocity- [22]

very-1.1.4.2 Demyelinating CMT (CMT1)

For the demyelinating form of CMT, genes are often

associated with Schwann cells and the myelin sheath

sunounding the axon Inheritance can be dominant,

recessive, or X-linked and the autosomal dominant form of

demyelinating CMT PMP22 (Peripheral myelin protein 22)

being the most common mutated gene, which resulted in CMT1Awith autosomal dominant inheritance fashion Patients with CMT1A present in the first two decades with a classical CMTphenotype,

starting with foot deformities and difficulty walking There is mainly distal involvementwith wasting and weakness of the muscles and sensoạ- loss [Li 2012 PMP22J.Autosomal recessive CMTl also known as CMT4, is relatively rare in the generalpopulation although this varies depending on the community The autosomal recessiveneuropathies tend to have an earlier onset and a more severe progression than theautosomal dominant varieties Except in consanguinity, they appear only in sibs or assimplex cases [23]

1.1.4.3 Axonal CMT(CMT2)

Axonal CMT also has both autosomal dominant (AD) and recessively inherited

fashion Several genes cause AD CMT2, but only a quarter of the patients receive a

molecular diagnosis There is no primary gene responsible for most cases in that PMP22

is the primary gene explaining the AD CMT1 phenotype From a genetic point of view,point mutations in the A/F.V2 gene account for 20% of patients and are the most

causing this disease's dominant variant, most of them ubiquitously expressed (Table1.2) The majority of these genes were not explicitly associated with die function of theaxon before mutations were discovered By discovering these mutations, criticalpathways were revealed that are necessary for maintaining axonal integrity

Axonal autosomal recessive neuropathies are very rare, and most cases found todate have been restricted to specific geographical areas or families In a recent study,

mutations in the histidine triad nucleotide-binding protein 1 (HĨNH) gene have been

found in 11% of AR peripheral neuropathy patients with neuronivotonia In patients with

AR CMT2 and neuromyotonia, this percentage went up to 76% Most of these familieswere eastern European and presented with the same homozygous mutation, suggesting afounder effect [25] [26] In North Africa, multiple families were found with mutations in

Lamin AC (LMN.-L), causing CMT2B1 LMNA mutations can cause various phenotypes,

ranging from peripheral neuropathies and cardiac disorders to lipodystrophy andpremature aging disorders More than ten different phenotypes have been shown to becaused by mutations in this gene, and many of them show overlapping clinical features[27] In 2013 a patient was reported with a mutation in the tripartite motifcontaining 2

{TRIM2) gene, which also encodes for an E3-Ubiquitin ligase [28] The loss of these

proteins in mouse models leads to neurodegeneration, indicating an essential role forthese proteins Figure 1.3 described the proposed pathomechanism of CMT and dHMN(reproduced from Rudnik-Schoneborn et al., 2020) [11]

1.1.4.4 Charcot-Marie-Tooth type 2S

CMT2S (OMIM 616155) is a recently discovered subtype of CMT2 in 2014 by

Ellen Cottenie et al Truncating and missense pathogenic variants on IGHMBP2 gene is

found to be the genetic defect leading to this condition CMT2S is a relatively pure form

of autosomal recessive axonal neuropathy characterized by onset in the first decade of

-c -ÍM Qỉ ugc V Hl

-■c -ÍM CỊỈ ugc V Hl

slowly progressive distal muscle weakness and atrophy affecting the lower and upperlimbs Patients have decreased reflexes and variable distal sensory impairment Thisgene has been found to be associated with Spinal Muscular Attophy with Respiratorydistress 1 or SMARD1 (also called distal Hereditary Motor Neuronopathy VI - dHMNVI), a severe medical condition with very’ early-onset and respiratory compromise (asdiscussed above) [8] A detailed comparison of the clinical manifestations of CMT2S andSMARD1 can be looked up in Table 1.3.

Cottenie et al (2014) reported 15 patients from 11

unrelated families with axonal neuropathy The families were of various ethnic origins, including English

European Serbian Korean Pakistani The age at onset was gait Some patients had a foot deformity, mainly pes

equinovarus Some also had hand weakness at onset, and almost all eventually developed significant hand

involvement Six patients used wheelchahs and most of the others required ankle-foot orthoses for walking Some

patients had scoliosis and mild proximal muscle weakness Physical examination

showed absent reflexes and variable distal sensor)’ impairment Three patients had anabnormal tongue shape, but otherwise, there was no bulbar or respirator)’ involvementElectrophysiologic studies were reported to be consistent with a mild motor and sensor)’axonal neuropathy with nene conduction velocities between 40 and 50 m s, althouijisome patients had absent responses on testing Sural nene biopsy from 1 patient showed

a moderate reduction in density of large myelinated fibers whereas tiny myelinatedfibers were well presented: ultrastructural analysis showed occasional activelydegenerating axonal profiles [8]

Cortenie et al (2014) identified biallelic mutations in the IGHMBP2 gene as the

cause- of this CMT2 type The pathogenic variants in the first family were found bywhole-exome sequencing; pathogenic variants in the remaining ten families were found

by targeted sequencing of a cohort of 85 families with recessive CNÍT2 Most of thepatients carried compound heterozygous mutations: many had a nonsense mutation inthe 5-prime region and a mutation in the last exon Patient fibroblasts and

lymphoblastoid cells showed IGHMBP2 protein levels lower than controls but higher

than those observed in patients with SMARD1 (dHMN VI), suggesting that the milderphenotype CMT2S may be related to residual protein levels [8]

Schottmann et al (2015) reported five patients from three unrelated families withCMT2S There was variable severity of the disorder One sib presented at age sixmonths with generalized hypotonia and had symptoms of respiratory insufficiency withdocumented diaphragmatic paralysis in one family He started using a wheelchau at agesix years Additional features included bladder and gastrointestinal dysfunction withachalasia His sister presented with delayed motor development and distal muscleweakness at age two She did not have respiratory symptoms but lost free independent

ambulation at age ten and had bladder and gastrointestinal dysfunction Three patientsfrom the other two families presented between 2 and 6 years of age with pes cavus andtoe walking but remained ambulator)' between ages 14 and 37 None had respirator)’symptoms All patients had absent reflexes and sensory symptoms, although sensorynerve action potentials were absent on electrophysiologic testing, consistent with asensorimotor neuropathy (29]

-c -ÍM Qỉ ugc V Hl

-■c -ÍM CỊỈ ugc V Hl

Table 1.2 Classification of Axonal CMT according to genotype.

Autosomal dominant

MTA TPaie 6

ATP production Autosomal recessive

1.2 The history of IGHMBP2 gene

The IGHMBP2 gene (Immunoglobulin Mu DNA Binding PTOtein 2) is

composed of 15 exons encoding a protein of 993 amino acids (Grohmann 2004) Fukita

et al (1993) used fluorescence in situ hybridization to map the human IGHMBP2 gene

to chromosome 1 Iql3.2-ql3.4 (Figure 1.4) [30] [9],

Figure 1.2 Location of IGH.XĨBP2 gene on chromosome 11

The IGHMBP2 protein also called Immunoglobulin S-p-binding protein 2 (Spbp-2) hasfour domains: an ATPase helicase domain, a single-stranded nucleic acid-binding R3Hdomain a DEXDc domain and an ANl-type zinc finger motif (Figure 1 5 A) (9) [18].[31] The IGHMBP2 protein is a member of the SF1 helicase SF1 helicases can bedivided into two classes SF1A and SF1B based on Ute direction of translocation, withSF1B further branched to Pifl-like (Pifl RecD2) and Upfl-like subfamily (fpfl.ighmbp2 Senataxin) [32] The Upfl-like family differs from die Pifl-like family as It canunwind both DNA and RNA duplexes in the 5' >3' dữection [33] The other twomembers of the Upfl-like family of SF1 helicases are Upfl an essential proteininvolved in nonsense-mediated mRNA decay (34) and Senataxin, implicated intranscription regulation (35)

Figure 1.3 Structural overview of hum an Ighmbpl protein (hlghmbp2) and the form of

human Ighmbpl that combining with an RNA (hlghmbpl-HNA).

(reproduced from Lun et al., 2012) Ị36Ị

Cardiac transcription factor-1 is a novel transcription factor that was first identified by McBride et al (1993) element located in the proximal enhancer of the atrial natriuretic factor (ANF: 108780) [37], Guenther et al

(2009) purified catalytically active recombinant IGHMBP2

and found that it functioned as an ATP-dependent 5-prime-

to-3-prime helicase that unwound RNA and DNA duplexes in

vitro JGHMBP2 localized predominantly to the cytoplasm of

neuronal and nonneuronal cells and associated with

ribosomes [18] Distal spinal muscular atrophy type-1

(DSMA1 Ố04320)-causing amino acid substitutions in IGHMBP2

did not affect ribosome binding, but they severely

impaired ATPase and helicase activity The authors proposed

that ĨGHMBP2 is functionally linked to translation and

that mutations in its helicase domain interfere with this function in DSMAỈ patients

De Planell-Saguer et al (2009) reported the biochemical characterization of

IGHMBP2 and the isolation of a modifier locus that rescued the phenotype and motor

neuron degeneration of nmd mice, the mouse model of SMARDỈ The authors mappedand localized the modifier locus to mouse chromosome 13 and generated a 166-kb BACtransgene derived from CAST EiJ mice containing tRNA genes and activator of basaltranscription-1 (Abtl) a protein-coding gene that is required for ribosome biogenesis

IGH.MBP2 associated physically with tRNAs particularly with tRNA-Tyr, which were

present in the modifier, and ỈGHMBP2 associated with the Abtl protein Transcription

factor HIC-220 kD (GTF3CI; 603246) an essential factor required for tRNAtranscription and the helicases reptin (RUVBL2; 604788) and pontin (RƯVBL1;603449) which function in transcription and ribosome biogenesis, were also part of7G//.W3P2-containing complexes De Planell-Saguer et al (2009) suggested that

IGHMBP2 may be a component of the translational machinery and that these

components may be genetically manipulated to suppress motor neuron degeneration[38]

In 20IS Viguire et al conducted a national multicenter observational retrospectivestudy to determine the prognosis of children with SXÍARD1 according to their

phenotype All known French pediatric cases with mutations identified on IGHMBP2

gene and respiratory symptoms were recorded and demonstrated in Figure 1.6 Theyconcluded that early onset of symptoms leading to specialist consultation before threemonths was associated with a significantly worse prognosis Among the six patientswho were still alive, all were tracheostomized [39]

Figure 1.4 IGHMBP2 genes and pathogenic variants

(reproduced front l iguier et al., 2018)

1.2.3.1 Spinal muscular atrophy with respiratory distress (SMARD1)

Grohmann et al (2001) demonstrated that autosomal

recessive distal spinal muscular atrophy type 1 (DSMA1; 604320) also known as spinal muscular atrophy with

respiratory distress (SMARD1) and distal hereditary motor

IGHMBP2 gene In DS MAI families, they detected three

recessive missense mutations (exons 5 11 and 12), two nonsense mutations (exons 2 and 5), one frameshift

mutation (exon 5) and one

splice donor site mutation (intron 13) The authors noted that mutations in mouse

IGHMBP2 are responsible for spinal muscular atrophy in the nmd mouse whose

phenotype resembles the DSMA1 phenotype Like the SA£V7 gene (600354),

IGHMBP2 colocalizes with the RNA processing machinery in both the cytoplasm and

the nucleus Grohmann et al (2001) concluded that IGHMBP2 is the second gene that

is found to be defective in spinal muscular atrophy and that IGHMBP2 and SMN sharestandard functions essential to motor neuron maintenance and integrity mammals [15]Among 29 infants with SMARD1 Grohmann et al (2003) identified 26 novel

mutations in the IGHMBP2 gene, including 14 missense, six nonsense, four frameshifis.

one in-frame deletion, and one frameshift insertion Pitt et al (2003) identified

mutations in the ỈGHMBP2 gene in 8 patients with severe infantile neuropathy with

diaphragmatic weakness and progressive axonal neuropathv The authors noted that thedisorder in their patients was slightly different from that described in classic SMARD1patients, most notably the absence of pathologic changes in the anterior horn in 1patient examined (9)

In 5 of 28 (18%) infants whose clinical course was consistent with SMARD 1

Maystadt et al (2004) identified nine novel mutations in the IGH.\IBP2 gene Seven of

the mutations occurred at highly conserved residues of the putative DXA helicasedomain of the protein Guenther et al (2007) identified 14 novel mutations in the

IGHMBP2 gene in 10 patients with SMARD1 All missense mutations altered conserved

residues within or adjacent to the putative DX’A helicase domain [40],

1.2.3.2 Cliarcot-.Marie-Tooth Disease, Axonal, Type 2S (CTM2S)

In 15 patients from 11 families with childhood-onset of autosoma 1 recessiveaxonal Charcot-Marie-Tooth disease type 2S (CMT2S; 616155), Cottenie et al (2014)

identified biallelic mutations in the IGHMBP2 gene The mutations in the first family

were found by whole-exome sequencing mutations in the remaining ten families werefound by targeted sequencing of a cohort of 85 families with recessive CMT2 Most ofthe patients carried compound heterozygous mutations many had a nonsense mutation

in the 5-prime region and a mutation in the last exon Patient fibroblasts and

Ivmphoblastoid cells showed IGHMBP2 protein levels lower than controls but higher

than those observed in patients with DSMA1 suggesting that the milder phenotype inCMT2S may be related to residual protein levels Functional studies of individualvariants were not performed, but Cottenie et al (2014) postulated a loss-of-functioneffect In 5 patients from 3 unrelated families with CMT2S, Schottmann et al (2015)

identified biallelic mutations in the IGHMBP2 gene [S], [29]

13 Prenatal Screening and Prenatal Diagnosis in Genetic diseases

1.3.1 Difference between prenatal screening and prenatal diagnosis

Prenatal testing may be offered to women during pregnancy to determine if the

fetus can be bom with a genetic condition or congenital disability Performing prenataltesting may help determine different options for the pregnancy or exceptionalmanagement of the pregnancy and delivery to improve the outlook for the baby Inorder to understand prenatal testing it is of great importance to distinguish betweenprenatal screening and prenatal diagnosis, the two concepts that made up the term

‘prenatal testing'

According to The American College of Obstetrics and Gynecology, Prenatalgenetic diagnostic testing is intended to determine, with as much certainty as possible,whether a specific genetic disorder or condition is present in the fetus In contrast,prenatal genetic screening is designed to assess whether a patient is at increased risk ofhaving a fetus affected by a genetic disorder Prenatal genetic diagnostic testing isintended to determine whether a specific genetic disorder or condition is present in thefetus with as much certainty as possible In contrast prenatal genetic screening isdesigied to assess whether a patient is at increased risk of having a fetus affected by agenetic disorder

Initial screening for congenital disabilities was

developed in the 1950s with ultrasound and has become

increasingly prominent in obstetric care Real-time prenatal diagnosis by

gray-evaluating pregnancies earlier in gestation Aims of ultrasonography includedetermining gestational age and fetal number, evaluation for malformations, testing offetal well-being, and assistance with invasive diagnostic and therapeutic procedures[41] Amniocentesis, the first available prenatal chromosomal diagnostic testing option,was first described in the 1950s [42] Amniocentesis has become increasingly safe and

is now used for several purposes, including genetic screening and infectious evaluationsChorionic villus sampling (CVS) is another diagnostic test and can be performed earlier

in gestation Subsequently, noninvasive tests, including serum analyte screening andcell-free DNA screening were developed for purposes of screening for geneticabnoimalities within a pregnancy

Screening tests can be performed in both the first and second trimesters ofpregnancy First-trimester screening involves an ultrasound examination and a sample

of the mother's blood, while second-trimester screening involves just the blood sample.The blood results and ultrasound results are then combined with maternal factors such

as age and weight to calculate the chance for certain chromosomal conditions in the

current pregnancy Screening results are usually available within a week and those whoreceive a positive result are offered diagnostic testing The detection rate for screeningtests varies by the type of test performed The only way to know whether or not adeveloping baby has a chromosomal condition is by performing a diagnostic test.

Specific diagnostic tests can determine with greater than 99.9 percent accuracywhether or not a developing baby has a chromosomal difference The nvo types ofdiagnostic tests are chorionic villus sampling (CVS) and amniocentesis Diagnostictests for specific genetic diseases must be specially requested These tests have differentsensitivity and specificity, depending on which test is ordered [43]

Most prenatal testing is intended for screening These tests include serumscreening, earner screening, and ultrasound the goals of these tests are to identifywomen with pregnancies at high risk of chromosomal abnormalities or congenitaldisabilities Although ultrasound can be diagnostic, such as open neural tube defectseruni screening is intended only to identify’ women with pregnancies at an increasedrisk Numerous options for serum screening are available with varying test criteria andtiming of employment (T able 1 4.)

Table 1.3 Overview of current serum screening options for aneuploidy

(reproduced from Carlson and I ora et aL, 2017) [44]

Screening Test

Gestational Age at Screening (weeks)

Analytes and or Measurements Obtained

DNA within the maternal serum

Cell-free DNA, commonly referred to as noninvasive prenatal screening, becamecommercially available in 2011 This relatively new technology involves collecting amaternal serum sample from which cell-free DNA fragments from the pregnancy are

isolated This cell-free DNA is primarily placental in origin and is released fromapoptotic trophoblasts Fetal fraction increases with gestational age but is reliably moresignificant than 10% as early as ten weeks gestation [45], [46] Notably, a fetal fractiongreater than 4% is required for reliable analysis [Fiorentino 2016] This screening testhas the highest available detection rate of all available screening tests for trisomy 21with a detection rate of 99% on a recently updated meta-analysis [Gil 2015], It isimportant to note that at present, cell-free DNA for aneuploidy screening is onlyrecommended by the ACOG for women with high pretest risk of aneuploidy: Maternalage greater than 35 years at delivery; Ultrasonographic findings indicating increasedaneuploidy risk: history of prior pregnancy affected by a trisomy, [47]

Ultrasound is now ubiquitous in pregnancy management

Nearly all women receive at least one ultrasonographic examination of their pregnancy during routine

obstetric care, and many receive more than one The primary function of ultrasound andobstetric care is for confirmation of dating and surveillance for congenital disabilities

Diagnostic testing allows patients to know with as much certainty as possiblewhether their pregnancy' may be affected by a particular genetic condition The mostcommon indication for diagnostic testing in the United States is the advanced maternalage or maternal age of 35 years or older on the estimated delivery' date Other commonindications include positive aneuploidv screening results, known family history ofgenetic disorders, or anomalies identified on ultrasound Although the ACOGrecommends diagnostic testing to be available to all women, regardless of maternal age.patients should be counseled before proceeding due to the risk of pregnancy loss

Chorionic villus sampling (CVS) remains the only diagnostic test available in thefirst trimester and allows for diagnostic analyses, including fluorescence in situhybridization (FISH), karyotype, microaưay molecular testing, and gene sequencing.CVS is performed between 11 and 13 week-gestation [48] CVS may be performed viaeither transcervical or transabdominal approach Via either approach, chorionic villi arecollected for genetic evaluation under ultrasound guidance without entering theamniotic sac CVS allows for earlier prenatal diagnosis, subsequently decreasing thetrine of uncertainty and allowing for earlier (and therefore, safer) pregnancytermination if desired However, a disadvantage of CVS is that approximately 1% to 2%

of CVS results may reflect confined placental mosaicism rather than actual fetalchromosomal abnormalities Confined placental mosaicism may increase the risk ofhaving a small-for-gestational-age infant (Baffero 2012] Pregnancy loss attributed toCVS is approximately 1 in 455 on the most recent estimates [4S], [49]

Amniocentesis is available in the second or third trimesters of pregnancy and may

be performed at any gestational age after 15 weeks In this technique, a sterile needle isinfroduced into the amniotic sac under ultrasound guidance, and amniotic fluid isobtained and sent for testing In addition to evaluation for genetic disordersamniocentesis mav also be used to evaluate for the presence of intra-amniotic or fetalinfection via culture or polymerase chain reaction or neural tube defects by measuring

r-u -ÍM Qỉ ugc V Hl

r-u -ÍM CỊỈ ugc V Hl

amniotic fluid alpha-fetoprotein and acetylcholinesterase Complications are relativelycommon at earlier gestational ages Pregnancy loss attributed to amniocentesis isapproximately 1 in 900 on most recent estimates [48], [49]

Preimplantation genetic diagnosis (PGD) is now widely

available and may even earlier detect chromosomal

abnormalities This procedure is performed after in vitro

fertilization (IVF) by manipulating the embryo to either remove a polar body or remove a single cell from the

blastocyst This procedure allows for detecting the

abnormality before embryo transfer so that only unaffectedembryos are transferred back It is different between PGD and PGS (preimplantation genetic screening) As for how it

is called PG s is a screening method, not a diagnostic PGD has a clear indication for those with prior identifiedmedical conditions That is PGD is currently applied for patients at high risk of transmitting a genetic

abnormality to their children, which includes all

dominant, and X-linked disorders) and carriers of balancedtranslocations, which are at high risk of implantation failure and recurrent abortions [50]

2.1 Time and location of the research

The research was conducted at Centre for Gene-Protein Research (Hanoi Medical University) from 2019 March to 2020 September

Location for amniocentesis: National Obstetrics and Gynecology HospitaL

04 patients with suspects of IGHMBP -2 related neuromuscular disorders and family members (parents, sibling)

Inclusive clinical criteria:

without a history of congenital heart disease or respirators’ disease

- Centrifuge and filtration Beckman (USA) Eppendorf (Germany)

■ Sanger Sequencing instrument ABI Prism 3100 Genetic Analyzer (USA)

2.4 Methods

2.4.1 Research protocol

Figure 2.1 Research protocoL

respiratory disưess due to prior medical condition or abnormalities (congenitaldiabetes, vitamin BI2 deficiency, autoimmune disease )