Claudia Gonzaga-Jauregui

Tamar Harel

Tomasz Gambin

Maria Kousi

Laurie B Griffin

Ludmila Francescatto

Burcak Ozes

Ender Karaca

Shalini Jhangiani

Matthew N Bainbridge

Kim S Lawson

Davut Pehlivan

Yuji Okamoto

Marjorie Withers

Pedro Mancias

Anne Slavotinek

Pamela J Reitnauer

Meryem T Goksungur

Michael Shy

Thomas O Crawford

Michel Koenig

Jason Willer

Brittany N Flores

Igor Pediaditrakis

Onder Us

Wojciech Wiszniewski

Yesim Parman

Anthony Antonellis

Donna M Muzny

Baylor-Hopkins Center

Mendelian Genomics

Nicholas Katsanis

Esra Battaloglu

Eric Boerwinkle

Richard A Gibbs

MD James R Lupski
email: jlupski@bcm.edu

Exome sequence analysis suggests genetic burden contributes to phenotypic variability and complex neuropathy

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ~45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy associated genes in subjects versus controls; confirmed in a second ethnically discrete neuropathy cohort, suggesting mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HMPVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.

Introduction

Charcot-Marie-Tooth (CMT) disease, first described clinically in 1886 [START_REF] Charcot | Sur une form particulie`re d'atrophie musculaire progressive, souvant familiale, debutant par les pieds et les jambes, et atteignant plus tard les mains[END_REF][START_REF] Tooth | The peroneal type of progressive muscular atrophy[END_REF], is a common hereditary peripheral neuropathy with an estimated prevalence of 1/1200 [START_REF] Braathen | Genetic epidemiology of Charcot-Marie-Tooth disease[END_REF] to 1/2500 [START_REF] Skre | Genetic and clinical aspects of Charcot-Marie-Tooth's disease[END_REF] individuals. The disease is characterized by distal symmetric polyneuropathy (DSP) with progressive muscle weakness and atrophy, and sensory loss. Two major clinical types are distinguished by electrophysiologic and neuropathologic studies and the type of cells (glia or neurons) primarily affected. CMT1 affects the glia-forming Schwann cells and presents with nerve conduction velocities (NCV) of <38 m/s; CMT2 affects the axons of neurons and usually presents with NCVs of >38m/s or slightly reduced motor NCVs but with diminished amplitudes. Other forms of CMT with additional clinical features have been described, including an intermediate form with overlapping demyelinating and axonal CMT features [START_REF] Nicholson | Intermediate forms of Charcot-Marie-Tooth neuropathy: a review[END_REF] and one in which CMT occurs in conjunction with glomerulonephritis [START_REF] Boyer | INF2 mutations in Charcot-Marie-Tooth disease with glomerulopathy[END_REF].

Observed inheritance patterns include: autosomal dominant, autosomal recessive and Xlinked (dominant and recessive) forms [START_REF] Lupski | Relation of hereditary pattern to clinical severity as illustrated by peroneal atrophy[END_REF][START_REF] Rossor | The distal hereditary motor neuropathies[END_REF]. Nevertheless, most patients present with apparent sporadic disease, attributable partially to the extreme clinical variability and age dependent penetrance of the phenotype. New mutation, however, is often the cause of sporadic CMT, with the de novo CMT1A duplication of 17p11.2 being responsible for 76-90% of sporadic cases [START_REF] Raeymaekers | Duplication in chromosome 17p11.2 in Charcot-Marie-Tooth neuropathy type 1a (CMT 1a)[END_REF][START_REF] Lupski | DNA duplication associated with Charcot-Marie-Tooth disease type 1A[END_REF][START_REF] Hoogendijk | De-novo mutation in hereditary motor and sensory neuropathy type I[END_REF][START_REF] Nelis | Estimation of the mutation frequencies in Charcot-Marie-Tooth disease type 1 and hereditary neuropathy with liability to pressure palsies: a European collaborative study[END_REF]. Locus-specific screening for mutations in known CMT genes concludes a molecular diagnosis for approximately 70-80% of patients [START_REF] Szigeti | Charcot-Marie-Tooth disease[END_REF][START_REF] Divincenzo | The allelic spectrum of Charcot-Marie-Tooth disease in over 17,000 individuals with neuropathy[END_REF]. More than 40 genes are known to be causative, but it has been estimated that 30-50 'CMT genes' remain to be discovered [START_REF] Braathen | Genetic epidemiology of Charcot-Marie-Tooth disease[END_REF][START_REF] Timmerman | Genetics of Charcot-Marie-Tooth (CMT) Disease within the Frame of the Human Genome Project Success[END_REF].

CMT1A [MIM #118220] is caused by a recurrent 1.4 Mb duplication that encompasses the dosage sensitive myelin gene PMP22 [START_REF] Lupski | DNA duplication associated with Charcot-Marie-Tooth disease type 1A[END_REF][START_REF] Hoogendijk | De-novo mutation in hereditary motor and sensory neuropathy type I[END_REF][START_REF] Patel | The gene for the peripheral myelin protein PMP-22 is a candidate for Charcot-Marie-Tooth disease type 1A[END_REF][START_REF] Lupski | Gene dosage is a mechanism for Charcot-Marie-Tooth disease type 1A[END_REF], an essential component of compact PNS myelin [START_REF] Li | The PMP22 Gene and Its Related Diseases[END_REF]. The reciprocal deletion of the identical 17p11.2 region causes hereditary neuropathy with liability to pressure palsies (HNPP) [MIM #162500] [START_REF] Chance | DNA deletion associated with hereditary neuropathy with liability to pressure palsies[END_REF][START_REF] Chance | Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17[END_REF]. A recent study of 17,000 patients with neuropathy established a molecular diagnosis in 18.5% of these; ~80% of molecular diagnoses were either duplication or deletion CNV of PMP22 [DiVincenzo, et al. 2014]. Point mutations and indels in PMP22 have also been found in patients with CMT1A or HNPP without duplication or deletion [Roa et al., 1993 (a); [START_REF] Nicholson | A frame shift mutation in the PMP22 gene in hereditary neuropathy with liability to pressure palsies[END_REF], and in the more severe early-onset phenotype of hypertrophic neuropathy of Dejerine-Sottas [MIM #145900] [START_REF] Dejerine | Sur la nevrite interstitielle, hypertrophique et progressive de l'enfance[END_REF]Roa et al., 1993 (a); Roa et al., 1993 (b); [START_REF] Li | The PMP22 Gene and Its Related Diseases[END_REF]. Additionally, non-recurrent and complex rearrangements can account for the missing heritability in CMT1A and HNPP, including upstream CNVs that do not include PMP22 coding sequence [START_REF] Zhang | Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: rare CNVs as a cause for missing heritability[END_REF][START_REF] Weterman | Copy number variation upstream of PMP22 in Charcot-Marie-Tooth disease[END_REF].

The second most common form of CMT is CMTX1 [MIM #302800] caused primarily by point mutations that occur in almost every amino acid of GJB1/connexin32 [START_REF] Kleopa | Molecular genetics of X-linked Charcot-Marie-Tooth disease[END_REF][START_REF] Scherer | X-linked Charcot-Marie-Tooth disease[END_REF]; gene deletions have also been described [Gonzaga-Jauregui et al., 2010]. GJB1 encodes a gap junction protein involved in the formation of connexon hemichannels that facilitate the communication and exchange of ions and other small molecules between Schwann cells and axons [START_REF] Scherer | X-linked Charcot-Marie-Tooth disease[END_REF].

The third most common cause of CMT, and the most common form of CMT2, are heterozygous mutations in MFN2 (CMT2A; [MIM #609260]) [START_REF] Othmane | Localization of a gene (CMT2A) for autosomal dominant Charcot-Marie-Tooth disease type 2 to chromosome 1p and evidence of genetic heterogeneity[END_REF][START_REF] Züchner | Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A[END_REF][START_REF] Verhoeven | MFN2 mutation distribution and genotype/phenotype correlation in Charcot-Marie-Tooth type 2[END_REF], essential for mitochondrial fusion and function [START_REF] Kijima | Mitochondrial GTPase mitofusin 2 mutation in Charcot-Marie-Tooth neuropathy type 2A[END_REF] and maintenance of mitochondrial morphology. Mutations in MFN2 lead to mitochondrial dysfunction due to mtDNA depletion [START_REF] Vielhaber | Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion[END_REF].

Mutations in GDAP1 cause a recessive form of CMT, which can be either demyelinating (CMT4A; [MIM #214400]) [START_REF] Cuesta | The gene encoding ganglioside-induced differentiation-associated protein 1 is mutated in axonal Charcot-Marie-Tooth type 4A disease[END_REF], axonal (CMT2K; [MIM #607831]) [START_REF] Nelis | Mutations in GDAP1: autosomal recessive CMT with demyelination and axonopathy[END_REF] or intermediate (CMTRIA;[MIM #608340]) [START_REF] Senderek | Mutations in the ganglioside-induced differentiation-associated protein-1 (GDAP1) gene in intermediate type autosomal recessive Charcot-Marie-Tooth neuropathy[END_REF] and have been reported to affect mitochondrial fission in Schwann cells and neurons [START_REF] Niemann | Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease[END_REF].

Known CMT genes encode proteins that span a wide range of functions, from GTPases (RAB7, DNM2), lipid phosphatases (FIG4, MTMR2), to structural myelin proteins (MPZ, PMP22) and gap junction channel components (GJB1). Cellular functions include myelin assembly (PMP22, MPZ, PRX, Cx32), membrane and endocytic trafficking (MTMR2, SBF2, FIG4, SH3TC2) and mitochondrial dynamics (MFN2, GDAP1) [START_REF] Niemann | Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease[END_REF][START_REF] Azzedine | Molecular genetics of charcot-marie-tooth disease: from genes to genomes[END_REF]. Another predominant contributing gene group is that of aminoacyl-tRNA synthetases, an essential class of enzymes that ligate amino acids onto cognate tRNA molecules [reviewed in [START_REF] Wallen | To charge or not to charge: mechanistic insights into neuropathy-associated tRNA synthetase mutations[END_REF].

Other complex forms of CMT2 (e.g. spinocerebellar ataxia with axonal neuropathy, SCAN1) have been associated with mutations in TDP1, important for DNA single strand break repair (SSBR) [START_REF] Mckinnon | DNA strand break repair and human genetic disease[END_REF][START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF]. Mutations in SETX, a helicase involved in transcriptional termination and RNA maturation, cause recessive ataxia ocular motor apraxia type 2 (AOA2; #606002) [START_REF] Moreira | Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2[END_REF] possibly due to transcriptional/translational defects [Anhelm et al., 2012], also disturbing DNA SSBR [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF]. SETX mutations have been associated with familial amyotrophic lateral sclerosis (ALS), susceptibility that recently was also associated with heterozygous FIG4 mutation carrier states [START_REF] Chow | Deleterious variants of FIG4, a phosphoinositide phosphatase, in patients with ALS[END_REF]. Substantial genetic and clinical heterogeneity of CMT neuropathy makes it challenging for molecular diagnosis by single gene and gene panel testing; the diagnostic utility of genomewide sequencing approaches has been demonstrated [START_REF] Lupski | Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy[END_REF][START_REF] Montenegro | Exome sequencing allows for rapid gene identification in a Charcot-Marie-Tooth family[END_REF][START_REF] Choi | Exome sequencing is an efficient tool for genetic screening of Charcot-Marie-Tooth disease[END_REF][START_REF] Lupski | Exome sequencing resolves apparent incidental findings and reveals further complexity of SH3TC2 variant alleles causing Charcot-Marie-Tooth neuropathy[END_REF]. We performed whole exome sequencing (WES) in a cohort of 40 patients with peripheral neuropathy from 37 unrelated families in whom extensive genetic evaluation had failed to identify a causative mutation or establish a molecular diagnosis (Table 1). Analysis of WES data was performed in two stages: a firstpass analysis that focused on known or novel variants in known CMT and related neuropathy genes, and a second stage analysis to search for rare variants in likely novel candidate genes (Supplementary Figure 1). Our rare variant analyses revealed potential neuropathy candidate 'disease genes'. Surprisingly, we uncovered evidence for a mutational burden in affected individuals versus a large sample of unrelated control individuals. We show experimentally that genetic interactions implicated by burden contribute to phenotypic variability and potentially to susceptibility to common neuropathies beyond the well characterized Mendelian forms.

Results

Known alleles in known neuropathy genes

We identified known disease-causing alleles in six of the 37 index patients (see Supplementary Information for detailed clinical information). Two represented phenotypic expansions of CMT2 caused by mutations in MFN2 (Figure 1A), where the clinical presentation made screening for MFN2 unlikely. One family showed two separate segregating causes of CMT [START_REF] Verny | Coincidence of two genetic forms of Charcot-Marie-Tooth disease in a single family[END_REF], one X-linked and the other caused by compound heterozygous mutations in MED25. A novel, likely disease causing allele was found in trans with the only known disease causing allele in this gene [START_REF] Leal | A second locus for an axonal form of autosomal recessive Charcot-Marie-Tooth disease maps to chromosome 19q13.3[END_REF][START_REF] Leal | Identification of the variant Ala335Val of MED25 as responsible for CMT2B2: molecular data, functional studies of the SH3 recognition motif and correlation between wild-type MED25 and PMP22 RNA levels in CMT1A animal models[END_REF]. In a proband with autosomal dominant neurosensory deafness and axonal neuropathy we found a recently reported mutation in MYH14 [START_REF] Choi | A complex phenotype of peripheral neuropathy, myopathy, hoarseness, and hearing loss is linked to an autosomal dominant mutation in MYH14[END_REF]. Finally, in a consanguineous family, we detected a 14kb homozygous deletion CNV encompassing exon 1 of ABHD12 segregating with the complex neuropathy phenotype observed in the proband and affected siblings [START_REF] Fiskerstrand | Mutations in ABHD12 cause the neurodegenerative disease PHARC: An inborn error of endocannabinoid metabolism[END_REF] (Supplementary Figure 2). An additional homozygous GDAP1 novel variant was also identified in some affected individuals of this family posing the possibility of an additive contribution from intragenic deletion CNV plus SNV variation.

Novel alleles in known neuropathy genes

Rare non-synonymous, frameshifting, or splicing variants were identified in known CMT/ neuropathy disease genes, illustrating the complexity that can underscore 'simple' mendelian conditions (see Supplementary Information for detailed clinical information). We identified a patient with mutations in both MFN2 and GDAP1, both of which are involved in mitochondrial dynamics. Concurrent mutations in these genes have been reported, suggesting the possibility of epistasis or modifying effects [START_REF] Cassereau | Simultaneous MFN2 and GDAP1 mutations cause major mitochondrial defects in a patient with CMT[END_REF][START_REF] Vital | A French family with Charcot-Marie-Tooth disease related to simultaneous heterozygous MFN2 and GDAP1 mutations[END_REF]. In a family with three generations affected by autosomal dominant intermediate CMT, we sequenced two individuals and identified a novel variant in YARS affecting a residue previously reported to be mutated in disease [START_REF] Jordanova | Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy[END_REF] (Figure 1B). Functional analyses revealed that the identified YARS allele is a functional hypomorph, unable to complement fully deletion of the endogenous yeast gene, TYS1, in growth complementation assays (Supplementary Figure 3), supporting a pathogenic role for this mutation in CMT. A male patient with Sotos syndrome [MIM #117550] due to NSD1 deletion, plus clinical neuropathy was found to carry several predicted deleterious variants in different CMT genes in addition to a novel potentially pathogenic variant in the X-linked AIFM1 gene [START_REF] Rinaldi | Cowchock syndrome is associated with a mutation in apoptosis-inducing factor[END_REF]. Compound heterozygous truncating mutations in SURF1 were identified in a proband with demyelinating CMT. Loss of function mutations in SURF1 were recently described in patients with autosomal recessive severe demyelinating neuropathy of childhood onset [START_REF] Echaniz-Laguna | SURF1 deficiency causes demyelinating Charcot-Marie-Tooth disease[END_REF], consistent with this patient's clinical and molecular findings.

Genetic and functional evidence for potential candidate CMT genes

We identified variant alleles implicating three potential new candidate neuropathy genes, PMP2, SPTLC3, and DNAJB5, in 3 different families. In a family with a clinical diagnosis of autosomal dominant demyelinating CMT1 neuropathy, we found a candidate missense variant in myelin protein P2, PMP2 (c.T128A; p.I43N) as the most likely disease causing variant. We confirmed this variant in the proband and his affected father, and its absence in both unaffected mother and sister (Figure 1D). PMP2 is a major stabilizing component of the myelin sheath that insulates the axons in the PNS [START_REF] Majava | Structural and functional characterization of human peripheral nervous system myelin protein P2[END_REF], but to date has not been associated with any genetic peripheral neuropathy. PMP2 is predominantly expressed in myelinating Schwann cells, with specific expression in sciatic nerve endoneurium and dorsal root ganglia [START_REF] Zenker | A role of peripheral myelin protein 2 in lipid homeostasis of myelinating Schwann cells[END_REF]. Homozygous knockout (Pmp2 -/-) mice have significantly reduced temporal motor nerve conduction velocities, although no major structural changes in the myelin sheath and peripheral nerves were observed [START_REF] Zenker | A role of peripheral myelin protein 2 in lipid homeostasis of myelinating Schwann cells[END_REF].

In vivo modeling experiments interrogated the potential impact of PMP2 loss of function and of this specific novel variant. Two orthologues exist in zebrafish; suppression of either using morpholino (MO) knockdown led to a motor neuron phenotype, including failure of the motor neuron axons to extend from the notochord, as well as pathfinding errors where the axons failed to innervate the myotomes appropriately (Figure 2A-B). These phenotypes could be rescued by co-injection of the MO with wild-type human PMP2; however contrary to wild-type, human mRNA carrying the variant identified in our proband failed to restore the MO induced phenotype (Figure 2A-D andE). Upon overexpression, wild-type human mRNA induced a phenotype similar to the one observed with MO alone in >50% of injected embryos, suggestive of a dosage-sensitive transcript, similar to PMP22. Overexpression of human mutant (p.I43N) PMP2 mRNA exacerbated the phenotype significantly (~20% increase; p=0.0003 Figure 2E-F); consistent with a dominant-negative mechanism of pathogenesis for this allele.

Of note, antibodies against PMP2 fragments were identified initially in experimental allergic neuritis, an autoimmune peripheral neuropathy in animals like rats and rabbits, and a model for Guillain-Barre syndrome (GBS) [START_REF] Ishaque | Allergic neuritis: phospholipid requirement for the disease-inducing conformation of the P2 protein[END_REF][START_REF] Ishaque | The complete amino acid sequence of the rabbit P2 protein[END_REF]. One of the main characteristics of GBS is the autoimmune attack to the peripheral nerves' myelin sheath causing demyelination. Antibodies against myelin protein zero (MPZ, P0) and most significantly to myelin protein 2 (PMP2, P2) have been detected in patients with GBS and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) characterized by primary demyelination and lymphocytic infiltration of the peripheral nerve [START_REF] Inglis | Antibody responses to peptides of peripheral nerve myelin proteins P0 and P2 in patients with inflammatory demyelinating neuropathy[END_REF]. Thus, discovery of this mutation in a CMT patient suggests a potential mechanistic link between auto-immune neuropathy and inherited neuropathy.

We identified a novel variant in SPTLC3 (c.T448C; p.W150R) changing a highly conserved residue and predicted to be damaging by bioinformatic algorithms in a patient; no parental samples were available. The proband presented with neuropathy with a marked sensory but no apparent autonomic involvement. SPTLC3 is the third subunit of the serine palmitoyltransferase enzyme (SPT) involved in the de novo biosynthesis of sphingolipids [START_REF] Hornemann | The SPTLC3 subunit of serine palmitoyltransferase generates short chain sphingoid bases[END_REF]. Heterozygous mutations in subunit 1 of SPT, SPTLC1, were first identified as the cause of hereditary sensory and autonomic neuropathy type 1A [HSAN1A; MIM #162400] [ [START_REF] Dawkins | Mutations in SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1, cause hereditary sensory neuropathy type I[END_REF]. Both genes encoding the additional subunits of SPT, SPTLC2 and SPTLC3, were screened for mutations in a cohort of typical HSAN patients. Heterozygous missense mutations were identified in SPTLC2 in a fraction of patients but no mutations were found in SPTLC3 [START_REF] Rotthier | Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I[END_REF]. Consistent with a neuropathy 'disease gene', suppression of the sptlc3 orthologue in zebrafish embryos showed motor neuron axon defects that phenocopied suppression of other known CMT genes (Figure 2H-I). The specific phenotype could be rescued by co-injection with SPTLC3 wild-type human mRNA (Figure 2H-L). Injection of human mRNA carrying the variant identified in the proband was unable to rescue the phenotype, supporting the contention that the missense variant represents a hypomorphic or possible loss of function allele (Figure 2H-L).

In a large family with an inheritance pattern consistent with an autosomal dominant myopathy/neuropathy, we identified 10 shared variants in three affected individuals, of which 9 did not segregate with the disease. A novel variant in DNAJB5 (c.C43T; p.P15S) was the only rare variant that co-segregated with the phenotype (Figure 1C). This rare variant was observed in four other independent individuals in our exome database of ~3,000 individuals; however no phenotypic information is available for these individuals. The variant is also present in the heterozygous state in a single individual in the Exome Aggregation Consortium (ExAC) compiled dataset (MAF=0.00004858). This DNAJB5 variant affects a highly conserved amino acid in the DnaJ domain of the protein. A homozygous mutation in DNAJB2 was identified in a large family segregating recessive distal hereditary motor neuropathy of early adulthood onset [START_REF] Blumen | A rare recessive distal hereditary motor neuropathy with HSJ1 chaperone mutation[END_REF]. Mutations in DNAJB6 have also been implicated in autosomal dominant myopathy [START_REF] Harms | Exome sequencing reveals DNAJB6 mutations in dominantly-inherited myopathy[END_REF][START_REF] Sarparanta | Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limbgirdle muscular dystrophy[END_REF] and have a dominant negative toxic effect increasing the stability of the cytoplasmic form of the protein and interfering with its chaperone function [START_REF] Sarparanta | Mutations affecting the cytoplasmic functions of the co-chaperone DNAJB6 cause limbgirdle muscular dystrophy[END_REF]. These three genes encode members of the HSP40/DNAJ family of molecular co-chaperones which protect proteins from irreversible aggregation during protein synthesis or molecular stress. Functional testing of this gene by MO knockdown in zebrafish showed abnormal peripheral nerve axonal architecture supporting a role of this gene in peripheral nerve pathophysiology but had no apparent effect on muscle architecture (Supplementary Figure 4). We propose DNAJB5 as a potential candidate for myopathy/neuropathy based on its relationship with previously reported genes involved in similar phenotypes; HSPB8 (HSP27) and HSPB1 (HSP22) are known genes associated with peripheral neuropathy [START_REF] Evgrafov | Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy[END_REF][START_REF] Irobi | Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy[END_REF].

Rare variant contributions to phenotypic manifestations -evidence for a mutation burden

WES of neuropathy patients often identified more than one rare variant in a neuropathy gene within a given personal genome (Table 2). As described above, we identified the predominant highly penetrant Mendelizing variants (HPMV) in multiple patients, as evidenced by co-segregation with disease or de novo appearance in sporadic neuropathy. However, we also identified potential contributing or modifying rare variants in other neuropathy associated genes (Figure 3). These latter rare variants are not likely the mutations predominantly responsible for trait manifestation because they are inherited from an unaffected parent or do not conform to Mendelian expectations (i.e. exceptions to cosegregation with neuropathy in the family). For example, we observed a higher than expected heterozygous carrier frequency of the reported MED25 (p.A335V) mutation in our cohort (10% of patients; MAF = 5.0%) compared to that observed in the NHLBI ESP study sample (65/6498 individuals; MAF=0.5% [P-value=0.001]), a group of 266 controls (2/266 individuals; MAF=0.375% [P-value=0.003]), and the ARIC European-American (ARIC-EA) study participants (80/ 5748 individuals; MAF=0.7%[P-value=0.003]). Although in 3 of 4 cases in our patient cohort there is no 'second hit' in MED25 to cause the CMT2B2 phenotype, we cannot discount the possibility of a second pathogenic non-coding variant not captured by WES or the potential contribution of this mutation in a mutational aggregation model to the overall phenotype of these patients.

Of note, we identified an average of 2.3 nonsynonymous rare variants per individual in 58 known neuropathy-associated genes in the entire patient cohort (37 samples) versus 1.3 nonsynonymous rare variants in 5748 ARIC-EA control individuals (P < 0.0001; Figure 4A). Cases with a definitive molecular diagnosis had an average of 2.9 variants per individual (including the HPMV) while the undetermined cases had an average of 1.8 variants per individual. After implementing a stringent filter where we subtracted the HPMV of each molecularly defined case, we still found an average of 1.8 variants in the CMT cohort vs. 1.3 in controls (P=0.007), similar to the average of mutations in only the cases without a yet definitive HMPV (Supplementary Figure 5). These data suggest that the mutation burden in CMT genes remains the same between patients with a known versus unknown HMPV and is significantly greater than the background load in unaffected controls.

As a further test of this mutational burden observation, we calculated repeatedly the average number of rare, nonsynonymous variants in the 58 neuropathy genes in 40 randomly selected individuals from the BHCMG_EU sample set compared to the 5748 ARIC_EA controls. Upon conclusion of 100 resamplings (with replacement), we only found three instances in which the p-value was lower than the p-value observed in our original US CMT (subtracting the HPMV) vs. ARIC_EA analysis;. These data reinforce the notion that the background mutation load in these 58 neuropathy genes is specific to the population of neuropathy patients.

To further investigate our observation of neuropathy gene mutation burden in neuropathy patients, we analyzed WES data from an independent cohort of 32 patients (30 families) from Turkey with a clinical diagnosis of CMT. When compared to population-matched unrelated Turkish controls, the Turkish neuropathy cohort had a mutation burden of 2.1 vs. 1.6 (P = 0.013) nonsynonymous rare variants per individual, lending further credence to the mutation burden hypothesis (Figure 4B, Supplementary Figure 5). The smaller difference in the number of rare variants per individual may also reflect a greater number of private variants in the Turkish population (particularly recessive alleles) or the contribution of consanguinity in this population.

Functional testing of the mutation burden hypothesis

We hypothesized that the 'mutation burden' observed in the CMT cohorts would be reflected in the functional consequences of CMT gene knockdown, and combinations thereof, in a zebrafish model. This functional assay evaluated the integrity and innervation of motor neuron axons along the body axis (Figure 5). A subset of genes was tested for potential genetic interactions and mutation burden effects on phenotype based on our initial cohort's observed mutation events. Specifically, we suppressed each of mfn2, gdap1, abhd12, med25, hspb1, and wnk1 separately and in pair-wise combinations of sub-effective doses and tested the functional consequences of the genetic interactions between the selected CMT genes. Consistent with our hypothesis, we observed increased severity in the phenotype of aberrant axon extension, branching, pathfinding, and morphology of peripheral neurons in our zebrafish model when we injected pairwise combinations of these genes (Figure 5). In each case, we observed likely multiplicative effects, although the magnitude of interaction was unique for each pairing. For example, sub-effective co-injection of MOs against mfn2 and gdap1, which by themselves gave no phenotype at the dose tested, yielded a milder exacerbated phenotype (Class I/II motor neuron pathology in 80-100% of embryos tested); whereas co-suppression of mfn2 and med25 yielded 80-100% affected embryos, with 1/3 of the embryos affected severely (Class III/IV). These data support the prediction of genetic interaction for loss of function events in bona fide CMT genes. To assess the specificity of our in vivo model we also tested for genetic interaction between GDAP1, a bona fide CMT driver, and 3 genes that have not been associated previously with peripheral neuropathy. Two of those are expressed in the CNS and cause other neuropathologies (SIX6: optic nerve atrophy [START_REF] Carnes | NEIGHBORHOOD Consortium Investigators. Discovery and functional annotation of SIX6 variants in primary open-angle glaucoma[END_REF]; RP1L1: retinal degeneration and cerebellar disorganization [Davidson et al., 2013]), and the third is expressed ubiquitously and causes VACTERL (ANKRD6; unpublished data). We injected sub-effective doses of each of the tested genes alone and also in pair-wise combinations (Supplementary Figure 6). Though RP1L1 yields a 20% increase in the percentage of embryos with abnormally formed peripheral neuronal axons when injected alone, we observed no exacerbation of the phenotype when each of those genes was suppressed in combination with GDAP1.

Discussion

Whole exome sequencing (WES) allows genome-wide assessment of SNV coding variation in the fraction of the human diploid genome that we can potentially interpret. However, even in genetic conditions with known associated genes, interpretation can be complicated by the presence of novel variants in more than one causative gene [START_REF] Yang | Clinical whole-exome sequencing for the diagnosis of mendelian disorders[END_REF][START_REF] Yang | Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing[END_REF]. Additionally, the contribution of variants in a multiplicity of genes for a single condition within an individual personal genome and how variation in these can contribute to or modify the phenotype has rarely been assessed.

We identified the apparent HPMV and likely primary disease driver of the neuropathy phenotype in 17/37 (45.9%) families studied and suggest a potential candidate gene for 3 additional families. We discovered a mutational burden of 2.3 damaging variants in CMT patients versus 1.3 in controls for the 58 neuropathy associated genes examined (P < 0.0001). After a highly stringent additional filter consisting of subtraction of the HPMV, neuropathy patients carry a mutation burden consisting of an average of 1.8 rare variants in neuropathy-related genes, as compared to an average of 1.3 rare variants in a control population (P=0.007). A mutation burden (P = 0.013) was replicated in a second, ethnically distinct CMT cohort in comparison to ethnically matched controls. This mutation burden may well influence the phenotype, contributing to the clinical heterogeneity and the spectrum of severity observed in the disease [START_REF] Haldane | The relative importance of principal and modifying genes in determining some human diseases[END_REF]. We explored this hypothesis in vivo examining phenotypic consequences of genetic interaction between select pairs of neuropathy genes. We observed increased severity of the phenotype in zebrafish consistent with potential additive and positive genetic interactions between neuropathy genes.

Our cohort has an intrinsic bias since individuals had previous extensive clinical and molecular screening for disease causing variation in the most common CMT genes prior to consideration for WES. As anticipated, we found a low frequency of known mutations as these samples were previously screened for such variants. We found variants in known CMT or neuropathy genes in 17 cases; including one (MFN2) showing phenotypic expansion in a CMT1 family. By expanding our candidate list to include additional neuropathy-associated genes, we achieved a 45.9% (17/37) mutation detection rate. Furthermore, we identified likely candidate genes PMP2, SPTLC3, and DNAJB5 in an additional 3 families potentially providing molecular insights into 20/37 (54.1%) of the families. We also provide functional evidence for the pathogenicity of the identified variants in PMP2 and SPTLC3 (Figure 2) and the effect of dnajb5 suppression on motor neurons (Supplementary Figure 4). However, conclusive proof for these genes representing bona fide 'neuropathy disease genes' will require the identification of pathogenic variants in additional patients.

Analysis of the WES data from this neuropathy cohort illustrates limitations of clinical phenotyping. Detailed phenotypic information is required for correlating potential disease causing variants to the clinical phenotype of patients. As illustrated in 12 of the study subjects, 8 from the initial cohort and 4 from the Turkish cohort originally referred for a presumptive clinical diagnosis of CMT, after a molecular diagnosis by WES and upon retrospective re-evaluation of clinical records, the broader spectrum of additional clinical features suggested other disorders associated with neuropathy. Moreover, these further refined phenotypes were consistent with the molecular findings from WES in each of the identified genes (Supplementary Table 1). The phenotype driven paradigm for clinical diagnosis is limited by the: i) presentation of the patient at the given time, ii) individual examiner and iii) underlying assumption of a singular unifying diagnosis; the latter potentially not applicable to either a mutation aggregation model or a mutation burden hypothesis.

In 29/40 (72.5%) patients we identified additional 'carrier status' mutations in other CMT or neuropathy associated genes besides the apparent HPMV (Table 2). These additional variants might contribute to the variability of expression of the clinical phenotype [START_REF] Haldane | The relative importance of principal and modifying genes in determining some human diseases[END_REF]. Furthermore, in the cases where specific HPMVs were not identified, novel loci potentially await to be recognized as main disease drivers (Supplementary Table 2), but the mutation burden may still contribute to variable expressivity of the neuropathy phenotype. It is possible that mutation burden and combinatorial effects of rare variants in genes that interact genetically in the same biological pathways, such as those of tRNA biogenesis, endocytic recycling or mitochondrial dynamics, modify the phenotype due to synergistic (exemplified by MFN2 and GDAP1 co-occurring mutations in the same patient) or counteracting effects [START_REF] Klassen | Exome sequencing of ion channel genes reveals complex profiles confounding personal risk assessment in epilepsy[END_REF][START_REF] Davis | The ciliopathies: a transitional model into systems biology of human genetic disease[END_REF]. Alternatively, or additionally, the cumulative mutation burden in genes dispersed across various biological pathways or 'networks' might interplay to destabilize or compensate the system and thus modulate the penetrance and/or expressivity of the overall phenotype. Although robust, the capacity of biological networks to buffer perturbations may be limited if various mutational events are coincident in a personal genome. Studies of the human disease network [START_REF] Goh | The human disease network[END_REF][START_REF] Hidalgo | A dynamic network approach for the study of human phenotypes[END_REF] at the genomic scale will likely contribute to our understanding of both disease and homeostatic states in human biology.

Genome-wide approaches have shown that rare variants are more common than previously thought [START_REF] Coventry | Deep resequencing reveals excess rare recent variants consistent with explosive population growth[END_REF][START_REF] Marth | The functional spectrum of low-frequency coding variation[END_REF]; a robust observation for both SNV and CNV disease associated alleles [START_REF] Boone | Deletions of recessive disease genes: CNV contribution to carrier states and disease-causing alleles[END_REF]. The overall phenotype of a given individual may to a greater extent represent contribution of either de novo or more recent and private mutational events with bigger effects on the whole system function, the 'driver' genes that occurred in the recent ancestors of the individual or clan [START_REF] Lupski | Clan genomics and the complex architecture of human disease[END_REF], rather than more distributed common variants shared in a population or throughout several populations. This mutation burden hypothesis and its role in clan genomics is further illustrated in CMT1A duplication families wherein a phenotypic outlier in the family is recognized when the duplication becomes a triplication [START_REF] Liu | Mechanism, Prevalence, and More Severe Neuropathy Phenotype of the Charcot-Marie-Tooth Type 1A Triplication[END_REF] or a CMT1A duplication is 'homozygosed' in a severe neuropathy patient born to heterozygous affected parents [START_REF] Lupski | DNA duplication associated with Charcot-Marie-Tooth disease type 1A[END_REF].

Interestingly, within peripheral neuropathies, several disorders once thought to be mostly caused by environmental factors, have been subsequently shown to have a genetic susceptibility component. A key example is provided by CNV at the PMP22 locus. The reciprocal to the CMT1A duplication, deletion of 17p11.2, causes Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) [START_REF] Chance | DNA deletion associated with hereditary neuropathy with liability to pressure palsies[END_REF]. Trait manifestation is usually associated with an environmental insult, trauma to a specific nerve and often those that come anatomically close to the surface (e.g. the ulnar nerve responsible for the 'funny bone' phenomena of numbness and tingling upon hitting the elbow). Locus-specific molecular studies revealed the majority of individuals that carry the HNPP deletion go undiagnosed [START_REF] Turner | Germline rates of de novo meiotic deletions and duplications causing several genomic disorders[END_REF] due to phenotypic variability or lack of clinical symptoms [START_REF] Kumar | Variability of presentation in hereditary neuropathy with liability to pressure palsy results in underrecognition[END_REF]. However, association of the deletion carrier status with susceptibility to developing carpal tunnel syndrome (CTS) has been documented [START_REF] Cruz-Martinez | Pediatric bilateral carpal tunnel syndrome as first manifestation of hereditary neuropathy with liability to pressure palsies (HNPP)[END_REF][START_REF] Potocki | DNA rearrangements on both homologues of chromosome 17 in a mildly delayed individual with a family history of autosomal dominant carpal tunnel syndrome[END_REF][START_REF] Del Colle | Hereditary neuropathy with liability to pressure palsies: electrophysiological and genetic study of a family with carpal tunnel syndrome as only clinical manifestation[END_REF]. Additionally, 24 of 51 patients diagnosed with multifocal neuropathies, not considered a genetic disease, were found to carry the HNPP deletion. Moreover, 37% of mutation positive subjects had no family history of neuropathy [START_REF] Tyson | Deletions of chromosome 17p11.2 in multifocal neuropathies[END_REF]. Consequently, haploinsufficiency of the dosage sensitive PMP22 gene, either by HNPP deletion (CNV) or loss of function point mutations [START_REF] Nicholson | A frame shift mutation in the PMP22 gene in hereditary neuropathy with liability to pressure palsies[END_REF][START_REF] Shy | T118M PMP22 mutation causes partial loss of function and HNPP-like neuropathy[END_REF], has been associated with susceptibility to milder forms of neuropathy. Furthermore, haploinsufficiency of the CMT SH3TC2 gene can also confer subclinical neuropathy phenotypes in heterozygous carriers, including subclinical axonopathy and median nerve mononeuropathy associated with susceptibility to CTS [START_REF] Lupski | Whole-genome sequencing in a patient with Charcot-Marie-Tooth neuropathy[END_REF].

From this perspective, our identification of a PMP2 variant, a gene whose product has been linked to experimental autoimmune neuropathy and both Guillain-Barre syndrome (GBS) and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), in one family suggests a potential genetic susceptibility to autoimmune neuropathy. Haploinsufficiency of other CMT or neuropathy genes can also contribute to susceptibility to multifactorial neuropathies. Moreover a recent study to survey possible underlying genetic contribution to developing chemotherapy induced peripheral neuropathy (CIPN) due to allelic variability in known CMT genes identified an association of PRX heterozygous variants in individuals that developed CIPN versus controls similarly exposed [START_REF] Beutler | Sequencing of Charcot-Marie-Tooth disease genes in a toxic polyneuropathy[END_REF]. Additionally, three common SNPs in ARHGEF10 were also associated to different outcomes of protection and susceptibility to CIPN in the same cohort [START_REF] Beutler | Sequencing of Charcot-Marie-Tooth disease genes in a toxic polyneuropathy[END_REF]. These findings support and highlight one of the main hypotheses from the present study; the mutation burden of carrier status, for neuropathy-associated rare variant recessive alleles, in clinically unaffected individuals can poise the organism to develop other types of complex neuropathies later in life upon gene-environment interactions (GxE). External insults, chemical or mechanical; other pathologic processes like diabetes or infection; or ageing with concomitant prolonged exposures, and/or reduced biological function of cells (e.g. SSBR, gene transcription, protein processing and folding, etc.) or functional units like the neuron can be the critical factor for the system to express the disease later in life. This might also be true for other traits thought to be complex and having a major environmental influence with a reduced genetic component that have been elusive to other approaches. Rather than single locus strong associations across populations, each individual with such a given complex disorder can carry a handful of rare/private variants in a variety of genes in their personal genome that are important for the development of the disease process and that through an oligogenic model confer susceptibility to the individual to develop the disorder upon additional factors such as diet, exposures, ageing, etc.

In summary, our studies of rare genomic variants in neuropathy identify known pathogenic alleles, novel variants in known disease genes, and further document phenotypic expansion for disease gene traits. We identified 3 potential novel candidate neuropathy 'disease genes' as supported by both genetic and functional studies. Moreover, we provide evidence that genome-wide studies and molecular diagnosis can further assist interpretation of a clinically based differential diagnosis. Of note, systematic analyses of genes implicated in neuropathy reveal a mutation burden in patients compared with unaffected control populations and zebrafish model organism studies show gene interactions for genes implicated by mutation burden in individual families. This mutation burden is consistent with the concept of clan genomics [START_REF] Lupski | Clan genomics and the complex architecture of human disease[END_REF] contributing significantly to both Mendelian and common/ complex disease trait manifestation.

Experimental Procedures

Samples

We performed WES through the Baylor-Hopkins Center for Mendelian Genomics (BHCMG). Written informed consent from all participating subjects was obtained for DNA and genetic analyses though a Baylor College of Medicine Institutional Review Board approved protocol, also approved by the BHCMG ELSI committee for inclusion into the BHCMG sequencing project. Some of these samples had been collected and stored over decades; thus, DNA of parents or other family members was not always available for additional testing and co-segregation analyses.

Exome sequencing

We performed whole-exome next-generation sequencing according to previously published methods [START_REF] Lupski | Exome sequencing resolves apparent incidental findings and reveals further complexity of SH3TC2 variant alleles causing Charcot-Marie-Tooth neuropathy[END_REF]; see Supplementary experimental methods for details], producing an average of 9.25 Gb of raw data per exome and achieving ~93.5x average depth of coverage (median coverage = 97x) per sample with >90% of the captured bases covered at 20x (Supplementary Table 3). Variant data generated will be released and deposited into the NCBI database of Genotypes and Phenotypes (dbGaP: http://www.ncbi.nlm.nih.gov/ gap) as part of the Centers for Mendelian Genomics research initiative.

Variant annotation pipeline

Variant calling from the aligned BAM files was performed using the ATLAS [START_REF] Shen | A SNP discovery method to assess variant allele probability from nextgeneration resequencing data[END_REF] and SAMtools suites [START_REF] Li | Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and SAMtools[END_REF]. Annotation was performed using Sacbe, an inhouse developed annotation pipeline [START_REF] Gonzaga-Jauregui | Mutations in VRK1 Associated With Complex Motor and Sensory Axonal Neuropathy Plus Microcephaly[END_REF] based on ANNOVAR [START_REF] Wang | ANNOVAR: Functional annotation of genetic variants from nextgeneration sequencing data[END_REF] and custom scripts (see Supplementary experimental methods for details).

Data analysis

We performed an initial analysis focusing on a list of 74 CMT and other neuropathy associated genes (Supplementary Table 4). Additionally, we interrogated a list of candidate CMT genes (Supplementary Table 5) based on first degree interactors of known CMT genes and performed a second pass analysis in those cases where we did not identify candidate mutations in CMT genes.

The number of rare (i.e., minor allele frequency of ≤1% in TGP, NHLBI ESP, and the European subset of NHLBI ESP) nonsynonymous variants in 58 well-established CMT genes (Supplementary Table 6) was computed for each sample of the neuropathy cohort and for 5748 Europeans from the ARIC (Atherosclerosis Risk in Communities study) cohort, a large population-based study of cardiovascular disease and its risk factors. The average number of rare nonsynonymous variants was then compared between the neuropathy and ARIC study samples using a non-parametric Mann-Whitney-Wilcoxon test. A permutation procedure with 100,000 iterations was performed to determine statistical significance. For the second CMT cohort of Turkish descent, a set of 472 Turkish controls was used that was sequenced and analyzed using identical protocols, platforms, and standards to those of the cases.

Functional experiments

See Supplementary experimental methods for details.

Medical Scientist Training Grant (GM07863), and an NIH F30 NRSA (NS092238). MS is supported by grants from MDA, CMT, NINDS/NCATS (U54NS065712) and from NINDS (R01NS075764). Suppression of pmp2 and sptlc3 in zebrafish causes defects in motor axon pathfinding and outgrowth. A-F. Lateral views of a control embryo, an embryo injected with pmp2 morpholino (MO) and embryos injected with pmp2 MO+PMP2_WT and pmp2 MO +PMP2_I43N, PMP2_WT and PMP2_I43N cocktails, respectively, at 2dpf (days post fertilization). Controls showed even spacing and normal branching of the motor neuron axons (A). In the pmp2 MO injected embryos the spacing of neuronal axons is perturbed by exiting the periphery but failing to extend (asterisks) or presenting pathfinding errors (arrows; B). Co-injection of pmp2 MO with human PMP2_WT resulted in restoration of the normal neuronal phenotype (C), but PMP2_I43N did not (D). Overexpression of human PMP2_WT causes mild pathfinding errors (E), suggesting dose sensitivity for PMP2. However, the human PMP2 mutant p.I43N, was significantly more severe than PMP2_WT when overexpressed (F) and had similar effects to suppression of pmp2 by MO knockdown. G. Percentage of normal versus abnormal embryos under the conditions being evaluated above. H-K. Wild type embryos (H) and sptlc3 morphants (I) in which secondary axons fail to migrate appropriately (white arrows). The phenotype induced by suppression of sptlc3 could be rescued by co-injection with SPTLC3_WT (J) but not SPTLC3_R150W (K). L. Quantification of normal embryos vs. embryos with motor neuron axon defects. For statistical analyses χ 2 -tests were performed. Neuron schematic of the localization or site of action of the main CMT/ neuropathy gene products. Legend on left shows patient identifier numbers and causative and possibly contributing mutations identified by WES. Full shapes correspond to rare presumed causative mutations deemed Highly Penetrant Mendelizing Variants (HMPVs); while empty shapes correspond to rare variants that may be contributing to the mutation burden in neuropathy patients. Each personal genome is distinguished by a unique color/shape. In bold are some of the canonical CMT genes. Rare variant distribution in studied individuals suggests high carrier frequency for rare alleles in neuropathy genes in exome sequenced neuropathy cohort. A different extended cohort of 5748 Europeans from the ARIC-EA study was observed to have a tendency towards zero or one rare variants in recessive neuropathy genes. Functional assessment of mutation burden hypothesis in a zebrafish model. First and second column panels show representative images of acetylated-tubulin (α-AcTub) staining of peripheral neurons in 2-day MO knockdown, single or in pair-wise combinations, zebrafish larvae. Third column panel shows qualitative assessment of morphant fish evaluated as defects in peripheral neuron axon extension, branching or pathfinding according to the scoring system developed. For pair-wise combinations, sub-effective concentrations of each of the gene-specific MOs were injected as shown in the graphs by the number of abnormal larvae in each category. However, when injected together increased severity in the phenotype was observed for all the pair-wise combinations, suggesting in vivo epistatic effects between these pairs of genes as observed in the α-AcTub fluorescence images and quantified in the graphs. Asterisks highlight some evidently affected axons. The scoring system used for assessing PNS defects in zebrafish was developed ad hoc and implemented here in order to best reflect the observations resulting from our experiments. Class I category refers to single axon defects; Class II category refers to two or more axons exhibiting defects with the presence of some normal axons; Class III category refers to generalized axonal defects; Class IV category refers to complete absence of axonal extension. previously published [START_REF] Pehlivan | Exome sequencing reveals homozygous TRIM2 mutation in a patient with early onset CMT and bilateral vocal cord paralysis[END_REF]

Proband

Figure 1 .

 1 Figure 1. Pedigrees of CMT/neuropathy patients and segregation of causative mutations. A. Pedigree showing de novo occurrence of the known p.V244M MFN2 mutation in proband. B. Dominant pedigree of a dominant intermediate form of CMT and segregation of the identified novel variant p.E196Q in YARS. Mutation was inherited to the affected proband and affected sister from the affected mother. C. Pedigree of a dominant form of CMT and segregation of the mutation in candidate gene PMP2 (p.I43N). The affected proband inherited the mutation from his affected father, while both unaffected mother and sister do

  Figure 2.

  Figure 3.

  Figure 4.

  Figure 5.

  abbreviations: CMT -Charcot-Marie-Tooth disease; DM -diabetes mellitus; DTRs -deep tendon reflexes; NCV -nerve conduction velocity; (-) = decreased/absent; NA -not available; NR -no response; nr -not reported; (T) -proband from Turkish cohort. Samples from the Turkish cohort are shaded in blue.*previously published[START_REF] Pehlivan | Exome sequencing reveals homozygous TRIM2 mutation in a patient with early onset CMT and bilateral vocal cord paralysis[END_REF]

Cell Rep. Author manuscript; available in PMC 2016 August 18.

Acknowledgements

This work was supported in part by US National Institute of Neurological Disorders and Stroke (NINDS) grant R01NS058529 to JRL; US National Human Genome Research Institute (NHGRI) grant National Heart, Lung, and Blood Institute (NHLBI) grant U54HG006542 to the Baylor-Hopkins Center for Mendelian Genomics and US National Human Genome Research Institute (NHGRI) grant U54HG003273 to RAG; by seed funding from the Center for Human Disease Modeling, Duke University and by P50 MH094268 to NK. AA is supported by a grant from the Muscular Dystrophy Association (MDA294479). WKW is supported by the Career Development Award K23NS078056 from NINDS. TH is supported by the T32 GM07526-37 Medical Genetics Research Fellowship Program. LBG was supported by the NIH Cellular and Molecular Biology Training Grant (GM007315), the NIH

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


Anastasiia Sheveleva

Christophe Finot

Temporal analogue of the Fresnel diffraction by a phase plate in linear and nonlinear optical fibers

Temporal analogue of the Fresnel diffraction by a phase plate in linear and nonlinear optical fibers

Introduction

⇒ we can observe spatial phenomena in the ultrafast optics ⇒ we consider Fresnel diffraction on phase plates

affiliation INTERPRETATION

Single phase transition

The phase transitions can be decomposed into two semi-infinite screens or into opaque stripe and slit, with one of the components additionally imprinting Δ𝜙 phase.

Output wave is the result of two-waves interference, where the intensity profile is defined by the phase difference (black lines) and is highly-dependent on Δ𝜙.

In all numerical (analytical) simulations we consider propagation in 10 km SMF-28 fiber with

𝛾 = 1.1 /W/km , 𝛽 2 = ±20 ps 2 /km

Double phase transition

LINEAR PROPAGATION

The waveforms are described by the Fresnel integrals (with the respective treatment and transitions to fiber optics).

-for the single phase jump one observes asymmetric profiles depending on Δ𝜙 and regime of dispersion -for the double phase jump the intensity profiles are always symmetric with respect to 𝑡 = 0, however it can be flipped with respect to Δ𝜙 = 𝜋 in case of the opposite dispersion regime

NONLINEAR PROPAGATION

Nonlinear Schrodinger equation (NLSE) :

𝜕𝑎(𝑡, 𝑧) 𝜕𝑧 + 𝑖 𝛽 2 2
𝜕 2 𝑎(𝑡, 𝑧) 𝜕𝑡 2 + 𝑖𝛾 |𝑎(𝑡, 𝑧)| 2 𝑎(𝑡, 𝑧) = 0 Existing intensity peaks trigger nonlinear effects leading to formation of coherent structures throughout the propagation :

• bright and dark solitons in case of the single phase jump • a Peregrine breather and a pair of dark solitons in case of the double phase jump

Conclusion

We study temporal intensity patterns emerging from diffraction by a phase plate.

-the results are interpreted in terms of interference of the diffracted waves on edge, stripe and slit -in case of purely linear evolution a complete analytical framework is provided -Kerr nonlinearity leads to the emergence of coherent structures


Anastasiia Sheveleva

Christophe Finot

All-fibered high-quality 28 GHz to 112 GHz pulse sources based on nonlinear compression of optical temporal besselons

All-fibered high-quality 28-GHz to 112 GHz pulse sources based on nonlinear compression of optical temporal besselons

Introduction

GOAL : generation of high-quality pulse trains at high repetition rates Besselon waveform [START_REF] Sheveleva | Temporal Optical Besselon Waves for High-Repetition Rate Picosecond Sources[END_REF] :

1. CW + temporal sinusoidal phase 𝜓 (𝑡) = exp(𝑖𝐴 𝑚 cos(𝜔 𝑚 𝑡))

⇒ Jacobi-Anger expansion :

𝜓 (𝑡) = ∑ ∞ -∞ 𝐽 𝑛 (𝐴 𝑚 ) exp(𝑖𝑛𝜔 𝑚 𝑡) 𝜔 𝑚 = 2𝜋𝑓 𝑚 -

Conclusion

-high repetition-rate source -simple setup (single phase modulator) -high-quality pulse trains at 14 GHz, 28 GHz, 56 GHz and 112 GHz

  repetition rate 𝐴 𝑚 -modulation amplitude (rad) 𝐽 𝑛 -Bessel function of the first kind of order 𝑛 ⇒ subsequent spectral lines have a 𝜋/2 phase shift 2. Triangular spectral phase to cancel out the phase offset [2] ⇒ Fourier transform limited waveform ⇒ high extinction ratio ⇒ full theoretical description available Besselon waveform generated at 14 GHz and 𝐴 𝑚 = 3.72 rad (FWHM = 7.2 ps, duty cycle = 0.2) red -theory, black -experiment phase for chirp compensation (red) -Filters used for multiplexing (black) HNLF : 𝐿 = 1 km, 𝛾 = 10 /W/km , 𝛽 2 = 0.89 ps 2 /km , 𝛼 = 0.5 dB/km Results at 28 GHz,56 GHz and 112 GHz 1. Phase-notch filter to multiplex the repetition rate before the nonlinear stage [3] Waveform at 28 GHz (FWHM = 7.2 ps , duty cycle = 0.2) Spectrum after NL propagation (extended over 1.4 THz) red -NLSE simulation, black -experiment,blue -autocorrelation of a Gaussian pulse After the chirp compensation : -high ER (more than 20 dB) -high stability -low duration (OSO and autocorrelation signal) 2. Attenuation of spectral lines of the NL-broadened spectrum to further multiplex red -NLSE simulation, black -