Genetic Spectrum of Inherited Neuropathies in India.

Background and
Objectives: Charcot-Marie-Tooth (CMT) disease is the commonest inherited neuromuscular disorder and has heterogeneous manifestations. Data regarding genetic basis of CMT from India is limited. This study aims to report the variations by using high throughput sequencing in Indian CMT cohort.
Methods: Fifty-five probands (M:F 29:26) with suspected inherited neuropathy underwent genetic testing (whole exome: 31, clinical exome: 17 and targeted panel: 7). Their clinical and genetic data were analysed.
Results: Age at onset ranged from infancy to 54 years. Clinical features included early-onset neuropathy (n=23), skeletal deformities (n=45), impaired vision (n=8), impaired hearing (n=6), facial palsy (n=8), thickened nerves (n=4), impaired cognition (n=5), seizures (n=5), pyramidal signs (n=7), ataxia (n=8) and vocal cord palsy, slow tongue movements and psychosis in one patient each. Twenty-eight patients had demyelinating electrophysiology. Abnormal visual and auditory evoked potentials were noted in 60.60% and 37.5% respectively. Sixty two variants were identified in 37 genes including variants of uncertain significance (n=34) and novel variants (n=45). Eleven patients had additional variations in genes implicated in CMTs/ other neurological disorders. Ten patients did not have variations in neuropathy associated genes, but had variations in genes implicated in other neurological disorders. In seven patients, no variations were detected.
Conclusion: In this single centre cohort study from India, genetic diagnosis could be established in 87% of patients with inherited neuropathy. The identified spectrum of genetic variations adds to the pool of existing data and provides a platform for validation studies in cell culture or animal model systems. Copyright:

INTRODUCTION

Inherited neuropathies are a heterogeneous group of diseases that predominantly affect the peripheral nerves but may be associated with other features such as spasticity, ataxia, global developmental delay, etc., resulting in complex syndromes.[1] These are slow-progressive disorders characterized by distal symmetrical weakness of the limbs, hypo/areflexia, and skeletal deformities that are more pronounced in the lower limbs.[23] They commonly begin in the first two decades of life, cause progressive disability, and impair quality of life.[45] They are broadly categorized on the basis of electrophysiological studies as: (a) demyelinating with predominant involvement of peripheral myelin wherein nerve conduction velocities are reduced, and (ii) axonal with predominant involvement of peripheral nerve axons wherein amplitudes of compound muscle action potentials are reduced with normal or slightly reduced nerve conduction velocities.

Among the inherited neuropathies, the hereditary motor and sensory neuropathies (HMSNs) or Charcot-Marie-Tooth diseases (CMTs) constitute the most frequent genetically determined neuromuscular disorders.[6] They are caused by mutations in genes encoding proteins involved in different peripheral nerve functions such as maintenance and compaction of myelin in Schwann cells, axonal transport, as well as mitochondrial metabolism and dynamics.[7] Progress in the field of genomics with the advent of the next generation sequencing (NGS) technology has led to the identification of a number of genes involved in various subtypes of CMTs in different ethnic groups across the globe.[38910] Identifying the molecular genetic abnormality establishes the diagnosis as well as aids in the treatment and reproductive planning.

India is a home to about one-sixth of the world population which is ethnically diverse, and has a distinct genetic landscape. Hospital based audits suggest that hereditary neuropathies account for 4.8% of all neuropathies.[11] Data on genetics of CMTs from India are rather limited.[1213] This study is aimed to identify variants in disease-associated genes using high throughput sequencing in a cohort of CMT from India.

PATIENTS AND METHODS

This study was carried out at the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India. Probands were recruited from a single neurology unit between March 2017 and February 2020, after obtaining written informed consent. They included subjects with chronic progressive sensorimotor neuropathy of suspected genetic etiology who did not have evidence of alternative etiologies such as acquired, autoimmune, or metabolic causes. Patients with duplication or point mutations in the PMP22 gene were published earlier and have not been included in the present study.[13] The clinical data included age, gender, symptom–duration, type of neuropathy, and sites of neuraxis affected. The functional/ambulatory status of the patients was objectively quantified using the CMT neuropathy score (CMTNS) and the modified Rankin Score (mRS).[1415] Nerve conduction studies were carried out using standard protocols at a laboratory temperature of 32–34°C (Neuropack S1 MEB-9400K, Nihon Kohden Corporation, Tokyo, Japan). Three motor nerves (median, ulnar, and common peroneal nerves) and three sensory nerves (median, ulnar, and sural nerves) were examined. Conduction blocks were considered if the ratio of the amplitudes of compound muscle action potentials following proximal and distal stimulation was <0.5, provided the distal amplitude was at least 20% of the lower limit of normal.[16] Recordings of evoked potentials to visual and auditory stimuli were carried out wherever possible. The study was approved by the Institute Ethics Committee of NIMHANS.

Genomic DNA was extracted using standard phenol–chloroform method from about 6 mL of peripheral blood collected in an ethylenediaminetetraacetic acid (EDTA)-coated vacutainer. Genetic analysis was carried out using NGS (whole exome: 31, clinical exome: 17, and targeted panel: 7). The libraries were prepared, followed by enrichment as per manufacturer's instruction for sequencing with 80-100X denotes the coverage of the exonic regions. The sequences obtained were aligned to the human reference genome (GRCh37/hg19) and analyzed using Sentieon for removing duplicates, recalibration, and re-alignment of indels. Sentieon haplotype caller was used to identify variants relevant to the clinical phenotype.[17] Common variants were filtered based on allele frequency in 1000Genome Phase 3, ExAC (v1.0), gnomAD (bv2.1), EVS, dbSNP (v151), and 1000 Japanese Genome.[18192021] The identified variants were interpreted based on the recommendations of American College of Medical Genetics and Genomics (ACMG).[22] The pathogenicity of the identified variants was predicted using multiple tools namely PolyPhen-2, sorting intolerant from tolerant (SIFT), and mutation taster. The data were entered in a predesigned proforma and incorporated into a Microsoft Excel Spreadsheet for analysis.

RESULTS

The current cohort comprised of 55 patients. The age at evaluation ranged from 2 to 72 years. The clinical and electrophysiological characteristics of patients are summarized in Table 1. In the present study, 62 variants were identified in 37 genes in these 55 probands. They included pathogenic/likely pathogenic variants (n = 28) and variants of uncertain significance (n = 34). There were 17 reported and 45 novel variants. Of these, eight patients had variants in more than one gene (MFN2+SBF, SH3TC2+AARS, SH3TC2+JPH 1, SH3TC2+UBQLN2, FGD4+WNK1, MARS+SBF2, MPZ+DNMT1, and GARS+GAN) implicated in the neuropathy phenotype. Three patients had additional variants in genes that so far have not been associated with neuropathy (DCTN1+BLK, IGHMBP2+SLC12A6, and MFN2+VPS13D). Ten patients did not have variants in neuropathy associated genes, but had variants in genes such as ATM, SETX, COX15, MPV17, OPA1, SACS, C10ORF2, APOB, and CDH23 which are implicated in other neurological disorders. In seven patients, no variants were detected [Tables 2 and 3]. In silico analysis showed that the detected variants were damaging [Supplementary Table 1].

Table 1

Clinical and electrophysiological characteristics of the current cohort of inherited neuropathy (N=55)

Parameter	Observed value
Males:Females	29:26
Age at evaluation	2-72 years.
Children (≤18 years)	25
Age at onset	Infancy to 54 years
Early onset neuropathy (onset ≤10 years)	23
Consanguineous parents	16
Positive family history	22
Developmental delay	9
Global delay	3
Motor delay	6
Skeletal deformities
Pes cavus	38
Hammer toes	30
Clawed fingers	19
Kyphoscoliosis	5
Pes planus	2
Dyschromatosis universalis hereditaria	1
Impaired vision	8
Ocular abnormalities	7
Optic atrophy	6
Retinitis pigmentosa	1
Cataract	1
Sensorineural hearing impairment	8
Facial palsy	8
Vocal cord palsy	1
Slow tongue movements	1
Thickened nerves	4
Intellectual disability or cognitive decline	5
Psychosis	1
Seizures	4
Pyramidal involvement	7
Ataxia	8
Cerebellar	4
Sensory	2
Mixed	2
Functional rating scales
CMT neuropathy score (mean±SD)	15.48±6.9
Modified Rankin score (mean±SD)	2.66±1.0
Electrophysiological tests
Demyelinating neuropathy (conduction velocity of ulnar nerve <38 m/sec)	26
Conduction blocks	7
Abnormal visual evoked potentials*	20**
Prolonged P100 latency	18
Absent waveforms	2
Abnormal brainstem auditory evoked responses*	11***
All waveforms absent	5
Only waves I and III present	1
Only wave V present	5

*Testing for visual evoked potentials and brainstem auditory evoked responses was carried out in 33 patients. **Seven of these patients with abnormal visual evoked potentials were symptomatic for impaired vision. ***Six of these patients with abnormal brainstem auditory evoked responses were symptomatic for impaired hearing

Table 2

Summary of genetic analysis of patients with inherited neuropathy included in the present cohort

Parameter	Number
Total number of genes identified with variations	37
Total number of variants	62
Missense	46
Intronic	1
Insertion	0
Deletions	1
Frameshift	8
Nonsense	4
Splice site	2
Zygosity
Homozygous (pathogenic/likely pathogenic/VUS)	23 (6/6/11)
Heterozygous (pathogenic/likely pathogenic/VUS)	36 (4/9/23)
Hemizygous (pathogenic/likely pathogenic/VUS)	3 (1/2/0)
Genes involved in patients with early onset neuropathy	APOB, C10ORF2, CDH23, COX6A1, FGD4, GDAP1, HK, HSPB8, IGHMBP2, JPH1, MTMR2, OPA1, SBF1, SLC12A6, UBQLN2, WNK1
Genes involved in patients with late onset neuropathy	AARS, ATM, BLK, COX15, DCTN1, DNMT1, GARS, LRSAM1, MARS, MME, MPV17, MPZ, NAGLU, SACS, SETX, VPS13D
Genes involved in patients with early and late onset neuropathy	GAN, GJB1, MFN2, SBF2, SH3TC2
Genes involved in patients with axonal neuropathy	APOB, BLK, C10ORF2, CDH23, COX15, COX6A1, DCTN1, GDAP1, LRSAM1, MME, MPV17, MTMR2, SBF1, VPS13D
Genes involved in patients with demyelinating neuropathy	AARS, ATM, DNMT1, FGD4, GAN, GARS, GJB1, HK, HSPB8, IGHMBP2, JPH1, MPZ, NAGLU, OPA1, SACS, SBF2, SH3TC2, SLC12A6, UBQLN2
Genes involved in patients with axonal and demyelinating neuropathy	MARS, MFN2, SETX, WNK1

Table 3

Genetic abnormalities identified in the present cohort of patients with inherited neuropathies (n=55)

Patient No	Clinical phenotype in addition to neuropathy	Demyelinating electrophysiology	Gene	Disease associated with genetic variant	Nucleotide change/Amino acid change/Zygosity	Inheritance	Classification	Reference	rsID
1	Facial weakness, pyramidal signs	No	MFN2	CMT2A	c. 281G>A/p.Arg94Gln/2Het	AD	Pathogenic	Reported[23]	rs28940291
2	Thickened nerves	No	MFN2	CMT2A	c. 605G>A/p.Gly202Asp/Het	AD	Likely pathogenic	Novel	Not available
		VPS13D	Spinocerebellar ataxia 4	c. 3005G>A/p.Gly1002Asp/Het	AR	VUS	Novel	Not available
3	Mild SNHL	No	MFN2	CMT2A	c. 281G>A/p.Arg94Gln/Het	AD	Pathogenic	Reported[23]	rs28940291
4	Seizures, intellectual disability, pyramidal signs	No	MFN2	CMT2A	c. 833T>C/p.Met278Thr/Het	AD	Likely pathogenic	Novel	Not available
5	-	No	MFN2	CMT2A	c. 371C>T/p.Ser124Phe/Homo	AR	Likely pathogenic	Novel	Not available
6	-	No	MFN2	CMT2A	c. 334G>A/p.Val112Met/Homo	AR	Likely pathogenic	Novel	rs757937208
7	Pyramidal signs	No	MFN2	CMT2A	c. 334G>A/p.Val112Met/Homo	AR	Likely pathogenic	Novel	rs757937208
8	-	No	MFN2	CMT2A	c. 310C>T/p.Arg104Trp/Het	AD	Pathogenic	Reported[24]	rs119103268
9	-	No	MFN2	CMT2A	c. 752C>G/p.Pro251Arg/Het	AD	Likely pathogenic	Reported[23]	rs1557525153
		SBF1	CMT 4B3	c. 2335C>G/p.Leu779Val/Het	AR	VUS	Novel	Not available
10	-	Yes	SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 1105C>T/p.Arg369Cys/Het	AR/AD	VUS	Novel	rs569974719
		AARS	CMT2	c. 2053G>A/p.Val685Met/Het	AD	VUS	Novel	Not available
11	-	Yes	SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 1412del/p.Leu471TrpfsTer53/Homo	AR	Pathogenic	Novel	Not available
		JPH1	CMT 2K	c. 803C>T/p.Pro268Leu/Het	AR/AD	VUS	Novel	rs756049890
12	Moderate SNHL, cerebellar and sensory ataxia	Yes	SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 3152G>A/p.Gly1051Glu/Homo	AR	VUS	Novel	Not available
		UBQLN2	ALS-15 with or without frontotemporal dementia	c. 1573C>T/p.Pro525Ser/Het	X-linked	VUS	Reported[25]	rs369947678
13	Facial weakness	Yes	SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 69del/p.Lys24ArgfsTer10/Homo	AR	Pathogenic	Novel	Not available
14	Thickened nerves	Yes	SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 3511C>T/p.Arg1171Cys/Het	AR/AD	Likely pathogenic	Reported[26]	rs759785462
		SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 2028G>C/p.Leu676Phe/Het	AR/AD	VUS	Novel	Not available
		SH3TC2	CMT4C, mild mononeuropathy of median nerve	c. 254A>T/p.Asp85Val/Het	AR/AD	VUS	Novel	Not available
15	-	Yes	GJB1	CMT1, HNPP	c. 548G>A/p.Arg183His/Het	X-linked	Pathogenic	Reported[27]	rs1555937233
16	-	Yes	GJB1	CMT1	c. 65G>A/p.Arg22Gln/Hemi	X-linked	Likely pathogenic	Reported[28]	rs1060501002
17	-	Yes	GJB1	CMT1	c. 217del/p.His73MetfsTer11/Hemi	X-linked	Pathogenic	Reported[29]	Not available
18	-	Yes	GJB1	CMT1	c. 77C>T/p.Ser26Leu/Hemi	X-linked	Likely pathogenic	Reported[30]	rs587777876
19	Facial weakness	Yes	FGD4	CMT4H	c. 1062_1063insT/p.Tyr355LeufsTer2/Homo	AR	Likely pathogenic	Novel	Not available
		WNK1	HSAN2, pseudohypoaldosteronism type 2	c. 7526C>A/p.Ser2509Tyr/Homo	AD/AR	VUS	Novel	Not available
20	-	Yes	WNK1	HSAN2, pseudohypoaldosteronism type 2	c. 2500G>A/p.Gly834Arg/Het	AD/AR	VUS	Novel	Not available
		WNK1	HSAN2, pseudohypoaldosteronism type 2	c. 4501+96C>A/Nil/Het	AD/AR	VUS	Novel	Not available
21	-	No	GDAP1	CMT 2K	c. 197C>G/p.Pro66Arg/Het	AR/AD	VUS	Novel	Not available
22	-	No	GDAP1	CMT 2K	c. 431C>T/p.Pro144Leu/Het	AR/AD	Likely pathogenic	Novel	rs786205591
23	Slow tongue movements, cerebellar ataxia	No	SETX	Spinocerebellar ataxia-1, spinocerebellar ataxia with axonal neuropathy 2, ataxia with oculomotor apraxia, Juvenile AlS-4	c. 3127_3128insA/p.Arg1043fs/Homo	AR	Likely pathogenic	Novel	Not available
24	Facial weakness	Yes	SETX	Spinocerebellar ataxia-1, spinocerebellar ataxia with axonal neuropathy 2, ataxia with oculomotor apraxia, juvenile AlS-4	c. 7195A>T/p.Ile2399Phe/Homo	AR	VUS	Novel	Not available
25	-	Yes	ATM	Ataxia telangiectasia	c. 4852C>T/p.Arg1618Ter/Het	AR	VUS	Novel	Not available
		ATM	Ataxia telangiectasia	c. 6899G>T/p.Trp2300Leu/Het	AR	VUS	Novel	Not available
26	OA, moderate SNHL, cerebellar ataxia, psychosis	No	MARS	CMT2U	c. 918_919del/p.Tyr307SerfsTer6/Het	AD	VUS	Novel	Not available
27	-	Yes	MARS	CMT2U	c. 2209C>T/p.Arg737Trp/Het	AD	Likely pathogenic	Reported[31]	Not available
		SBF2	CMT4B	c. 3110G>A/p.Arg1037His/Homo	AR	VUS	Novel	Not available
28	Cognitive decline, thickened nerves	Yes	SBF2	CMT4B	c. 5345_5354del/p.Asp1782ValfsTer10/Homo	AR	Pathogenic	Novel	Not available
29	-	No	MME	CMT2T	c. 1270G>A/p.Gly424Arg/Homo	AR	VUS	Novel	Not available
30	-	Yes	MPZ	CMT1B, CMT2J, CMT 2I, CMT with hearing loss and pupillary abnormalities	c. 223G>T/p.Asp75Tyr/Het	AD	VUS	Novel	Not available
31	Facial weakness, moderate SNHL	Yes	MPZ	CMT 1B	c. 207_212delGCCCGA/p.Pro70_Glu71del/Het	AD	VUS	Novel	Not available
		DNMT1	HSAN 1E, AD cerebellar ataxia, CMT	c. 1018G>A/p.Ala340Thr/Het	AD	VUS	Novel	rs529074384
32	Cognitive decline, thickened nerves	No	LRSAM1	CMT 2P	c. 2120C>T/p.Pro707Leu/Het	AD	Likely pathogenic	Novel	rs797044913
		LRSAM1	CMT 2P	c. 49C>T/p.Arg17Cys/Het	AD	VUS	Novel	rs368646898
33	-	No	DCTN1	dHMN VIIB, Perry syndrome, ALS	c. 3746C>T/p.Thr1249Ile/Het	AD	Likely pathogenic	Reported[32]	rs72466496
		BLK	Maturity onset diabetes of young type 11, systemic scleroderma, rheumatoid arthritis	c. 211G>A/p.Ala71Thr/Het	AD	VUS	Reported[33]	rs55758736
34	-	No	COX15	Leigh syndrome due to cytochrome c oxidase deficiency	c. 520G>A/p.Gly174Ser/Homo	AR	Likely pathogenic	Novel	rs763842058
35	Pyramidal signs	No	COX6A1	Intermediate CMTD	c. 247-7_247-3del (3’ proximal splice site)/Homo	AR	Pathogenic	Reported[34]	rs587777783
36	Facial weakness	Yes	GARS	CMT 2D, dHMN VA	c. 1172G>A/p.Arg391His/Het	AD	VUS	Novel	rs370057212
		GAN	Giant axonal neuropathy 1	c. 944C>G/p.Pro315Arg/Het	AR	VUS	Reported[35]	rs144486241
37	-	Yes	GAN	Giant axonal neuropathy 1	c. 444C>G/p.His148Gln/Homo	AR	VUS	Novel	Not available
38	Intellectual disability, facial weakness, seizures	Yes	HK	Russe type of HMSN	c. 19C>T/p.Arg7Ter/Homo	AR	Pathogenic	Novel	rs779250530
39	Sensory ataxia, pyramidal signs	No	IGHMBP2	CMT2S, AR distal SMA1, dHMN	c. 1523C>T/p.Ser508Leu/Homo	AR	Likely pathogenic	Reported[36]	rs754465226
		SLC12A6	Hartnup disease, Andermann syndrome	c. 1625T>C/p.Ile542Thr/Homo	AR	VUS	Novel	Not available
40	-	No	MPV17	Mitochondrial DNA depletion syndrome-6	c. 280G>T/p.Gly94Trp/Homo	AR	VUS	Novel	Not available
41	Facial weakness, vocal cord palsy	No	MTMR2	CMT4B	c. 484C>T/p.Arg162Ter/Homo	AR	Pathogenic	Novel	rs756723587
42	-	Yes	NAGLU	CMT2V	c. 325C>T/p.Arg109Cys/Het	AD	VUS	Novel	Not available
43	OA, RP, cataract, cerebellar ataxia	No	OPA1	Optic atrophy plus syndrome	c. 1045C>T/p.Arg349ter/Het	AD	Pathogenic	Novel	Not available
44	Mild SNHL	Yes	SACS	Spastic ataxia of Charlevoix-Saguenay type	c. 8980C>T/p.Pro2994Ser/Homo	AR	VUS	Novel	Not available
45	Severe SNHL, sensory ataxia	No	C10ORF2	Mitochondrial DNA depletion syndrome-7	c. 876delT/p.Ala293ProfsTer33/Het	AR	Likely pathogenic	Novel	rs772683219
46	Pyramidal signs	Yes	HSPB8	CMT 2L, dHMN	c. 71C>T/p.Ser24Phe/Het	AD	VUS	Novel	rs781475312
47	OA, seizures, cerebellar ataxia	No	APOB	Familial hypercholesterolemia, familial hypobetalipoproteinemia	c. 13441G>A/p.Ala4481Thr/Homo	AR	VUS	Reported[37]	rs1801695
48	OA, severe SNHL	No	CDH23	Usher syndrome, non-syndromic hearing loss, age-related hearing loss	c. 1589-7C>T/Homo	AR	Novel	Not available
49	-	Yes	Nil
50	-	No	Nil
51	Pyramidal signs	No	Nil
52	-	Yes	Nil
53	Mild SNHL, seizures	Yes	Nil
54	OA	No	Nil
55	Cognitive decline, OA, cerebellar and sensory ataxia	No	Nil

AD: Autosomal dominant, ALS: Amyotrophic lateral sclerosis, AR: Autosomal recessive, dHMN: distal hereditary motor neuronopathy, Hemi: hemizygous, Het: heterozygous, HNPP: hereditary europathy with liability to pressure Palsy, Homo: Homozygous, HSAN: Hereditary sensory autonomic neuropathy

Supplementary Table 1

In silico prediction of the pathogenicity of the identified variants

Patient No	Gene	Nucleotide change/Amino acid change	Zygosity	ACMG Classification	SIFT	Polyphen2	LRT	Mutation taster
1	MFN2	c. 281G>A/p.Arg94Gln	Het	Pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
2	MFN2	c. 605G>A/p.Gly202Asp	Het	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
	VPS13D	c. 3005G>A/p.Gly1002Asp	Het	VUS	Affect protein function	-	-	Damaging
3	MFN2	c. 281G>A/p.Arg94Gln	Het	Pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
4	MFN2	c. 833T>C/p.Met278Thr	Het	Likely pathogenic	Tolerated	Benign	Damaging	Damaging
5	MFN2	c. 371C>T/p.Ser124Phe	Homo	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
6	MFN2	c. 334G>A/p.Val112Met	Homo	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
7	MFN2	c. 334G>A/p.Val112Met	Homo	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
8	MFN2	c. 310C>T/p.Arg104Trp	Het	Pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
9	MFN2	c. 752C>G/p.Pro251Arg	Het	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
	SBF1	c. 2335C>G/p.Leu779Val	Het	VUS	Affect protein function	Possibly damaging	Damaging	Damaging
10	SH3TC2	c. 1105C>T/p.Arg369Cys	Het	VUS	Tolerated	Possibly damaging	-	Damaging
	AARS	c. 2053G>A/p.Val685Met	Het	VUS	Tolerated	-	Damaging	Damaging
11	SH3TC2	c. 1412del/p.Leu471TrpfsTer53	Homo	Pathogenic	-	-	-	Damaging
	JPH 1	c. 803C>T/p.Pro268Leu	Het	VUS	Tolerated	Benign	Damaging	Damaging
12	SH3TC2	c. 3152G>A/p.Gly1051Glu	Homo	VUS	Affect protein function	Probably damaging	Damaging	Damaging
	UBQLN2	c. 1573C>T/p.Pro525Ser	Het	VUS	Tolerated	Benign	-	Damaging
13	SH3TC2	c. 69del/p.Lys24ArgfsTer10	Homo	Pathogenic	-	-	-	Damaging
14	SH3TC2	c. 3511C>T/p.Arg1171Cys	Het	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
	SH3TC2	c. 2028G>C/p.Leu676Phe	Het	VUS	Affect protein function	Probably damaging	Damaging	Damaging
	SH3TC2	c. 254A>T/p.Asp85Val	Het	VUS	Tolerated	Possibly damaging	-	Damaging
15	GJB1	c. 548G>A/p.Arg183His	Het	Pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
16	GJB1	c. 65G>A/p.Arg22Gln	Hemi	Likely pathogenic	Affect protein function	Possibly damaging	Damaging	Damaging
17	GJB1	c. 217del/p.His73MetfsTer11	Hemi	Pathogenic	-	-	-	Damaging
18	GJB1	c. 77C>T/p.Ser26Leu	Hemi	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
19	FGD4	c. 1062_1063insT/p.Tyr355LeufsTer2	Homo	Likely pathogenic	-	-	-	-
	WNK1	c. 7526C>A/p.Ser2509Tyr	Homo	VUS	-	-	-	-
20	WNK1	c. 2500G>A/p.Gly834Arg	Het	VUS	-	Damaging	-	-
	WNK1	c. 4501+96C>A/Nil	Het	VUS	-	-	-	-
21	GDAP1	c. 197C>G/p.Pro66Arg	Het	VUS	Affect protein function	-	-	-
22	GDAP1	c. 431C>T/p.Pro144Leu	Het	Likely pathogenic	Affect protein function	-	-	-
23	SETX	c. 3127_3128insA/p.Arg1043fs	Homo	Likely pathogenic	-	-	-	-
24	SETX	c. 7195A>T/p.Ile2399Phe	Homo	VUS	Affect protein function	Possibly damaging	Damaging	Damaging
25	ATM	c. 4852C>T/p.Arg1618Ter	Het	VUS	-	-	-	-
	ATM	c. 6899G>T/p.Trp2300Leu	Het	VUS	Tolerated	-	-	-
26	MARS	c. 918_919del/p.Tyr307SerfsTer6	Het	VUS	-	-	-	Damaging
27	MARS	c. 2209C>T/p.Arg737Trp	Het	Likely pathogenic	Affect protein function	Possibly damaging	Damaging	Damaging
	SBF2	c. 3110G>A/p.Arg1037His	Homo	VUS	Affect protein function	Possibly damaging	Damaging	Damaging
28	SBF2	c. 5345_5354del/p.Asp1782ValfsTer10	Homo	Pathogenic	-	-	-	Damaging
29	MME	c. 1270G>A/p.Gly424Arg	Homo	VUS	Affect protein function	Probably damaging	Damaging	Damaging
30	MPZ	c. 223G>T/p.Asp75Tyr	Het	VUS	Affect protein function	Probably damaging	Damaging	Damaging
31	MPZ	c. 207_212delGCCCGA/p.Pro70_Glu71del	Het	VUS	-	-	-	-
	DNMT1	c. 1018G>A/p.Ala340Thr	Het	VUS	Tolerated	Benign	Benign	Benign
32	LRSAM1	c. 2120C>T/p.Pro707Leu	Het	Likely pathogenic	Affect protein function	Probably damaging	Damaging	Damaging
	LRSAM1	c. 49C>T/p.Arg17Cys	Het	VUS	Affect protein function	Probably damaging	Damaging	Damaging
33	DCTN1	c. 3746C>T/p.Thr1249Ile	Het	Likely pathogenic	Affect protein function	-	-	Damaging
	BLK	c. 211G>A/p.Ala71Thr	Het	VUS	-	Possibly damaging	-	-
34	COX15	c. 520G>A/p.Gly174Ser	Homo	Likely pathogenic	Tolerated	Probably damaging	Damaging	Damaging
35	COX6A1	c. 247-7_247-3del (3’ proximal splice site)	Homo	Pathogenic	-	-	-	Damaging
36	GARS	c. 1172G>A/p.Arg391His	Het	VUS	Tolerated	Possibly damaging	Damaging	Damaging
	GAN	c. 944C>G/p.Pro315Arg	Het	VUS	Tolerated	Benign	-	Damaging
37	GAN	c. 444C>G/p.His148Gln	Homo	VUS	Tolerated	Probably damaging	Damaging	Damaging
38	HK	c. 19C>T/p.Arg7Ter	Homo	Pathogenic	-	-	-	Damaging
39	IGHMBP2	c. 1523C>T/p.Ser508Leu	Homo	Likely pathogenic	Affect protein function	Damaging	Damaging	Damaging
	SLC12A6	c. 1625T>C/p.Ile542Thr	Homo	VUS	Tolerated	Possibly damaging	-	-
40	MPV17	c. 280G>T/p.Gly94Trp	Homo	VUS	Tolerated	Possibly damaging	-	Damaging
41	MTMR2	c. 484C>T/p.Arg162Ter	Homo	Pathogenic	-	-	-	Damaging
42	NAGLU	c. 325C>T/p.Arg109Cys	Het	VUS	-	Possibly damaging	-	Damaging
43	OPA1	c. 1045C>T/p.Arg349ter	Het	Pathogenic	-	-	-	Damaging
44	SACS	c. 8980C>T/p.Pro2994Ser	Homo	VUS	-	Possibly damaging	Damaging	Damaging
45	C10ORF2	c. 876delT/p.Ala293ProfsTer33	Het	Likely pathogenic	-	-	-	-
46	HSPB8	c. 71C>T/p.Ser24Phe	Het	VUS	Tolerated	Possibly damaging	Damaging	Damaging
47	APOB	c. 13441G>A/p.Ala4481Thr	Homo	VUS	-	Benign	-	-
48	CDH23	c. 1589-7C>T	Homo	-	-	-	-	-

DISCUSSION

This study led to the identification of pathogenic/likely pathogenic variants in 87.3% cases, in addition to a number of novel variants as well as variants of uncertain significance (VUS). However, in other cohorts the detection rates ranged from 24% to 87% based on high throughput sequencing.[383940] The clinical, demographic, and genetic features in various cohorts of CMT are compared with the present study [Supplementary Table 2].[87] Previous studies have shown that variants in PMP22, GJB1, MPZ and MFN2 genes account for vast majority of the CMTs.[941] Variants in other genes though individually rare, constitute a large number, with nearly 100 genes being implicated in the pathogenesis of CMT.[42] Given the large number of genes implicated in CMT, it may be difficult to precisely pinpoint the genetic abnormality based on the phenotype, because of significant overlapping clinical features. For instance, vocal cord palsies have been described in both axonal and demyelinating neuropathies due to MFN2, GDAP1, TRPV4, SH3TC2, and MTMR2 mutations.[43] Sensorineural hearing loss (SNHL) has also been reported in demyelinating and axonal CMT due to PMP22, GJB1, MPZ, PRPS1, and SH3TC2 mutations, among others.[44454647] We used NGS to identify the genetic basis in patients of Indian origin with suspected inherited neuropathies in whom the PMP22 variants had been excluded. The most frequent abnormality in the present study was in the MFN2 gene (all pathogenic/likely pathogenic), which is similar to that noted in the previous studies. MFN2 is reported to be the commonest cause of axonal CMT followed by MORC2.[48] In the present study, no variants in MORC2 were identified. Variants in SH3TC2 were the second most frequent abnormality in the present cohort (pathogenic/likely pathogenic = 3, VUS = 4), which is reported to be the commonest cause of recessively inherited demyelinating CMT.[4950] Variants in GJB1 and MPZ were identified in four and two patients, respectively. An interesting finding in the present cohort is that a proportion of patients had conduction blocks on electrophysiological testing. Classically, demyelination in CMT is considered to be uniform and conduction block is generally not expected in electrophysiological testing. However, while in the “pre-genetic” era it was believed that inherited neuropathies have uniform reduction in conduction parameters, there is growing evidence that some of the CMTs may exhibit non-uniform conduction abnormalities as well as conduction blocks. In fact, conduction block and non-uniform slowing can form the basis for targeted genetic testing (e.g., X-linked CMT).[51]

Supplementary Table 2

Epidemiological, demographic, clinical and genetic features in various cohorts of Charcot Marie Tooth disease

Author/Year	Country	Cohort Number	M: F ratio	Age at onset (years)	Sporadic/Familial	Genetic Test	Genetic Diagnosis	Reported/Novel	VUS
Mostacciuolo et al., 2001[53]	Italian	172 cases	-	-	35/104	Targeted gene sequencing	PMP22 duplication=98/170 cases MPZ point mutations: 4 variants PMP22 missense mutations: 2 variants C×32: 12 variants	Reported=14 Novel=4	-
Sivera et al., 2013[54]	Spanish	438 cases	-	-	-	Targeted gene sequencing	Total yield=365/438 (83.3%) Most common: PMP22 duplication=184 cases Point mutations- GJB1=56 cases GDAP1=42 cases SH3TC2=27 cases MPZ=19 cases NDRG1, HSPB1=7 cases each MFN2=6 cases HK1=5 cases	Novel=17	-
Manganelli et al., 2014[57]	Italian	197 cases	-	-	47/101	Targeted gene sequencing	Total yield=148/197 (75.1%) PMP22=107 GJB1=14 GDAP1=8 MPZ=7 SH3TC2=3 MFN2=2	Novel=12	-
Hoyer et al., 2015[56]	Norway	103 cases	48:55	-	-	MLPA, Targeted gene sequencing	Total yield=35/103 (33.9%) Point mutations=28 cases Copy number variations=7 cases	-	10
Antoniadi et al., 2015[86]	-	448 cases	-	-	-	Targeted gene sequencing	Total yield=137/448 (30.5%) 195 variants in 31 genes for 137 patients AD inheritance=93/137 AR inheritance=32/137 X-linked inheritance=9/137	Reported=107 Novel=88	215
Drew et al., 2015[58]	-	110 cases	-	-	2/108	Whole exome sequencing	Total yield=21/110 (19.09%) HMN=2 cases HMNP=4 cases CMT2=10 cases CMT1=1 case CMTX=4 cases	Reported=9 Novel=12	- Rudnik-
Schöneborn et al., 2016[59]	German	1330 cases	-	Varied from early infantile (<2 years), to late adult (>50 years)	894/436	MLPA, Targeted gene sequencing	AD/X-linked inheritance (axonal) = 108/340 (31.8%) AD/X-linked inheritance (demyelinating) = 275/674 (40.8%) Autosomal recessive inheritance (axonal) = 15/340 (4.4%) Autosomal recessive inheritance (demyelinating) = 38/674 (5.6%)	-
Nam et al., 2016[60]	Korean	78 families	-	1-49 years	-	Hexaplex microsatellite PCR, Targeted panel sequencing	Total yield=17/78 (21.7%) GJB1=6 variants MPZ=2 variants SH3TC2=1 pair of compound heterozygous PMP22, MARS, MFN2, SPTLC2, DCTN1=1 variant each	Reported=7 Novel=8	-
Li et al., 2016[61]	Chinese	22 cases	17:5	Childhood to 46 years	-	MLPA, Targeted gene sequencing	PMP22 duplication=8/22 Possible pathogenic variants: 11/22	Reported=7 Novel=3	-
Sun et al., 2017[10]	Chinese Han	106 patients, NGS done on 82	57:25	Mean 30±15 years	86/20	NGS	PMP22 duplication=10 patients GJB1 mutation=9 patients PMP22 deletion=2 patients MFN2 mutation=2 patients NEFL, SH3TC2, HSPB1, PRX=1 patient each	Reported=15(single base exchange) Reported Copy number variation=2(PMP22 duplication, PMP22 deletion) Novel=6(single base exchange)	-
Dohrn et al., 2017[62]	German	612 cases	294:318	-	289/217	MLPA and NGS	Total yield=121/612 (19.7%) PMP22=16.4% GJB1=10.7% MPZ & SH3TC2=9.9% MFN2=8.3%	Reported=121 cases Novel=34 variants	201
Bacquet et al., 2018[38]	French	179 cases (123 prospective and 56 retrospective)	-	-	-	Targeted panel of genes causing inherited disorders	Total yield=49/123 (39.8%) CMT1=19/28 CMT2=27/64 dHMN=5/11 HSAN=5/9 Internediate CMT=4/8	Reported=26 Novel=52	17
Milley et al., 2018[9]	Hunagrian and Roma	531 cases	289:242	First decade to seventh decade of life	142/148	MLPA, qPCR, targeted gene sequencing	Total yield=59.9% CMT1=276 CMT2=42	Reported=30 Novel=6	-
Hartley et al., 2018[24]	Canadian	50 index patients and 23 affected/unaffected family members	-	Adult onset: 34 cases Pediatric onset: 16 cases	11/39	NGS	Total yield=12/50 (24%) HMSN=8/34 HMN=4/11 HSN or HSAN=0/5	-	11
Hoebeke et al., 2018[39]	French	75 cases from 59 different families	1.8:1	Mean: 4.1 years	21/54	MLPA, Targeted gene sequencing, Targeted inherited disease panel for NGS	PMP22 duplication=46/75 MFN2 mutations=11/75 Other genes=18/75	Reported=12 Novel=10	-
Yoshimura et al., 2018[63]	Japanese	1005 patients	-	-	570/413	NGS (CMT panel)	Total yield=301/1005 (30%)	-	-
Khadilkar et al., 2017[12]	Indian	22 patients	19:3	-	18/4	NGS	Total yield=13/22 (63.07%)	-	3
Hsu et al., 2019[64]	Taiwanese	427 patients	248:179	Mean=23.8±17.4 years Range=1-72 years	177250	Real time fluorescent PCR for PMP22, direct sequencing of PMP22, GJB1, MPZ, MFN2, NEFL, AARS, HSPB1, GDAP1. NGS	Total yield=312/427 (73.1%) Demyelinating CMT=266/315 (84.4%) Axonal CMT=46/112 (41.1%)	Reported=69 Novel=12	-
Cortese et al., 2020[8]	UK and US	220 cases	136:84	-	111/109-	NGS	Demyelinating CMT=30/41 axonal or intermediate=32/143 dHMN =/21 HSN=2/15	Novel=30	98
Taghizadeh et al., 2020[65]	Iran	58 patients	-	Mean=13 years Range=4 months to 63 years	-	NGS (WES)	Total yield=27/58 (46.6%)	Reported=16 Novel=11	-
Xie et al., 2021[87]	Chinese	435 families	268:167	1-60 years	221/214	MLPA for PMP22, NGS (CMT panel)	Total yield=304/435 (70%)	Reported=140 Novel=20	66
Current cohort	Indian	55 patients	29:26	Infancy to 54 years	Sporadic=33 Familial=22	NGS (WES=31, CES=17, Neurology/CMT panel=7)	28/55 (50.9%)	Reported=17 Novel=45	33

dHMN: distal Hereditary motor neuronopathy, HSAN: Hereditary sensory and autonomic neuropathy, HSN: Hereditary sensory neuropathy, MLPA: Multiplex ligation-dependent probe amplification

In the present study, in addition to the common genes (GJB1, MPZ, and MFN2), variants were identified in a number of genes implicated in various cellular functions such as growth and differentiation (SBF1, DCTN1), endocytosis (SH3TC2, LRSAM1), tRNA synthetases (AARS, MARS, GARS), intracellular calcium homeostasis (JPH 1), ubiquitin-proteasome system (UBQLN2, GAN), actin cytoskeleton regulation (FGD4), DNA repair (SETX, ATM), transcriptional regulation (IGHMBP2, DNMT1), protein homeostasis (MME, HSPB8), mitochondrial function including dynamics and maintenance (MPV17, COX6A1, COX15, VPS13, OPA1, C10ORF2), and ion transport (SLC12A6). Besides these, alterations in endoplasmic reticulum structure (TFG, ATL1), membrane or vesicle trafficking (LITAF, SBF1, DNM2, FIG4), myelin structural organization (PRX), axonal cytoskeleton maintenance (NEFL, NEFH), and axonal transport (KIF1, DCTN1, SPG11) have been reported from other cohorts of HMSN.[6667]

The CMTs may follow autosomal or X-linked, dominant or recessive pattern of inheritance. Homozygous or compound heterozygous variants in the “dominantly” inherited genes resulting in recessive CMTs with early onset and more severe disability have been reported. For example, MFN2 is usually linked to autosomal dominant (AD) inheritance but autosomal recessive (AR, homozygous or compound heterozygous variants) pattern is also reported where the proband inherits one mutation from each parent. The heterozygous parents can be asymptomatic or may manifest with late-onset milder phenotype, in contrast to early-onset severe phenotype in the proband bearing two mutations.[6869] We also report two homozygous variants in MFN2 in three subjects with early-onset neuropathy [Patients 5–7, Table 3]. In contrast to compound heterozygous variants that occur in trans, distantly spaced double variants in cis have also been reported uncommonly in MFN2.[7071] Likewise, LRSAM1 variants are dominantly inherited and are associated with CMT2 phenotype with onset in the second decade of life and moderate disability.[72] In the present study, patient 32 had early-onset neuropathy with severe disability and two heterozygous variants in LRSAM1. The presence of two variants might have contributed to the increased disease severity. This phenomenon may be comparable to severe phenotypes associated with homozygous/compound heterozygous mutations in MFN2.

The present study identified variants in multiple genes in 11 patients. There are a few reports highlighting co-occurrence of variants of multiple genes in the same individual with CMT.[737475767778] Such variants are often inherited not only from heterozygous carrier parents, but can also occur de novo. High throughput sequencing permits unbiased analysis of several genes and helps in identifying all the variants which could have been missed in sequential analysis. Traditional sequencing of genes in tandem does not extend testing for other genes once a genetic variant that explains the phenotype is identified. Using NGS one can identify multiple genetic variants in different combinations: (a) more than one CMT-associated genes, or (b) CMT-associated gene(s) implicated in other neurodegenerative disorders. Two variants identified in different neuropathy-related genes may cause mild phenotype when they occur in isolation, but when present together may have additive effect on severity of symptoms by causing pathology at different sites. The non-neuropathy related genetic variants may act as modifier when present together with another gene known to cause neuropathy.[77] The most common cause of demyelinating CMT, that is, PMP22 duplication has been reported with additional dose of PMP22 (triplication) resulting in more severe clinical phenotype.[7980] There are reports on PMP22 mutation along with other related genes like LITAF, SMN2, DCTN1, GJB1, FSHD, and ABCD1 [Supplementary Table 3].[73747576] Coexistence of variants in MFN2 and GDAP1 in axonal CMT has also been reported. The two variants act in a synergistic manner resulting in major mitochondrial defects as each gene is involved in mitochondrial bioenergetics either for adenosine triphosphate (ATP) production or respiratory chain complex I activity.[81828384] Apart from PMP22 and MFN2, there are selected reports on other gene combinations such as JPH 1/GDAP1 and EGR2/GJB1.[557785] Increased genetic “burden” arising from this combination of genetic mutations may contribute to phenotypic variability including age at onset and disease severity.[78] Based on the available literature, we hypothesize that the co-occurence of multiple genetic variants may have impacted the clinical phenotype including the severity in the present cohort. However, we did not establish the synergy between the multiple variants and their impact on the phenotypes by using in vitro studies or animal models. This is a limitation of the present study.

Supplementary Table 3

Impact of mutations in multiple genes on neuropathy phenotype

Author/year	Gene combination	Number of subjects	Impact on phenotype
Kim et al., 2015[70]	PMP22 triplication	1 case	Proband: severely affected: triplication, mildly affected family members: duplication
Liu et al., 2014[71]	PMP22 triplication	Triplication is caused due to Lenovo mutation from maternal origin and results in severe phenotype as compared to usual duplication
Meggouh et al., 2005[50]	PMP22 and LITAF	1 case	PMPP22 causes inefficient protein folding and variations in LITAF may hamper protein degradation pathway, altogether affecting the clearance of misfolded protein. Modifier genes can play role for pathogenesis of disease
Fernández et al., 2016[51]	SMN2 and PMP22	1 case	Clinical phenotype suggestive of SMN, Elder brother was diagnosed with CMT
Hodapp et al., 2006[66]	PMP22 and GJB1, PMP22 and DCTN1, PMP22 and ABCD1	3 families	Presence of two gene variants resulted in cumulative effect on severity of symptoms, and individual variant itself was correlated with respective function in peripheral nerve
Chung et al., 2005[76]	EGR2 (R359W) and GJB1 (V136A)	Screening: 125 CMT families, described: 1 family; 5 members	Proband carrying both the mutations had severe phenotype while father having mutation only in EGR2 gene presented with mild phenotype. The difference in clinical presentation might be either due to genetic modifier in EGR2 (mild phenotype) or cumulative effect of both the mutations (severe phenotype)
Kim et al., 2010[77]	DMPK (CTG repeats) and GJB1 (R149Q)	1 family	DMPK inherited from father (80 fold as compared to 220 fold in proband), mother was found normal for the repeats, GJB1: mother carried heterozygous mutation but this variant was absent in father
Vital et al., 2012[75]	MFN2 (V160fs) and GDAP1 (R120W)	1 family	Mother and father both heterozygous carriers of one mutation each. Proband and her daughter have both the variants. Synergistic effect of two mild variants resulted in severe phenotype observed in the second generation (proband) and her daughter as well
Kostera-Pruszczyk et al., 2014[74]	MFN2 (T236M) and GDAP1 (H123R)	1 case	MFN2 variant: inherited from maternal line (mother and paternal grandfather carriers); results in impaired mitochondrial energy coupling and GDAP1 variant: denovo; impairs mitochondrial transmembrane potential. Individual variants presents with mild phenotypes as reported earlier in literature, but this combination resulted in severe phenotype
Anghelescu et al., 2017[72]	MFN2 (P201L) and GDAP1 (E222K)	1 case	Proband: both GDAP1 and MFN2 mutations. Father and paternal grandmother carriers of GDAP1 variant, MFN2 variant was not found in any of the family members examined. Father and paternal grandmother had mild phenotypic presentation, de novo MFN2 mutation or co-existence of MFN2 and GDAP1 may explain the severity of disease in the proband
Cassereau et al., 2011[73]	MFN2 (R468H) and GDAP1 (Q163X)	1 family	MFN2 variant results in mild phenotype, as ATP production remains normal in this case even when there is defect in energy coupling, but GDAP1 variant results in decreased ATP production because of impairment in Complex I activity. Simultaneous MFN2 and GDAP1 mutations cause major mitochondrial defects in a patient with CMT. The synergistic effect of these two mutations prove to be deleterious and hence explaining the severity of phenotype
Pla-Martín et al., 2015[68]	JPH 1 and GDAP1	-	GDAP1 variant (R120W) and JPH 1 variant (R213P) collectively mimics the phenotype of GDAP1 knock-down cells as they both are involved in calcium homeostasis
Schreiber et al., 2013[67]	FSHD and PMP22	1 case	Overlap of two phenotypes

The present study identified a number of novel variants and VUS which require to be validated for confirming their pathogenicity. Previous studies have also reported a large number of VUS, ranging from 10 to as high as 215 in a single cohort.[5686] Reporting of VUS is dependent on the ACMG guidelines. Validating individual variants in various genes may not be an efficient approach given the low frequency of individual genetic variants other than PMP22, MPZ, GJB, and MFN2 genes. Due to the prevailing high levels of genetic heterogeneity, narrowing down to common cellular pathways through network biology approach and forming “disease modules” may prove to be more useful in understanding the pathobiology even in patients who are “negative” for genetic abnormalities by whole exome sequencing (WES). Various genes reported in the context of hereditary neuropathies act on interconnected pathways and share common proteins to carry out the overlapping biological functions. The peripheral nervous tissue being highly metabolically active needs constant maintenance of a pool of proteins and other molecular interactors. Mutations in any one of the associated genes resulting in abnormal protein can have a cascading effect on the protein interactome and may fail to maintain the cellular homeostasis. This effect is propagated along the nerve function adding to disease pathology. These networks of proteins and their molecular partners can be exploited further to understand the disease pathogenesis and further translated for drug development and therapeutics.[52]

In conclusion, we report the NGS findings in a fairly large cohort of patients with inherited neuropathies from India and highlight the spectrum of genetic abnormalities. This study brings out a number of novel variants and VUS. Establishing an accurate genetic diagnosis is important not only for genetic counseling but also in the perspective of including patients for upcoming therapeutic trials. NGS identified variants in several genes, including those that have pathobiological significance in neuropathy and other non-neuropathic disorders. The functional validation of novel variants and the impact of their interactions with other molecular partners remain to be established in future studies.

Financial support and sponsorship

The study was funded by a grant to Dr Madhu Nagappa from the Indian Council of Medical Research (BMS/TF/Trans-Neuro/2014-3389/July-15/16/KA/Govt dated 25th July 2016).

Conflicts of interest

There are no conflicts of interest.