the study was approved by the institutional review board of the charit ( ea1/222/08 ) . parents provided written informed consent for all aspects of the study according to the declaration of helsinki . we performed initial autozygosity mapping followed by whole - exome sequencing ( wes ) of a consanguineous family with 1 affected patient ( figure 1 ) and 2 healthy siblings . for autozygosity mapping , we investigated the index patient ( patient ii:03 , figure 2a ) and 2 healthy siblings ( ii:01 and ii:02 ) using the affymetrix 6.0 genechip snp array and performed subsequent analysis using homozygositymapper2012 software ( http://www.homozygositymapper.org ) to identify genomic regions that were homozygous solely in the index patient . subsequent wes was performed on the index patient after capture with the sureselect v4 kit ( agilent technologies , santa clara , ca ) and sequencing on a hiseq 2000 machine ( illumina , san diego , ca ) , yielding 82 mio 2  101 bp paired - end reads that were aligned to the human grch37.p11 ( hg19/ensembl 72 ) genomic sequence . the mean coverage was 112 , and 87.9% of the captured sequences were covered more than 10 . after fine adjustment , the raw alignments were called for deviations from the reference in all coding exons 20 bp flanking regions . each of these variants was submitted to mutationtaster2 software to search for potentially pathogenic variants . these variants were further filtered for a recessive inheritance model ( the variant allele occurs in homozygous state in > 20 healthy individuals from the exac database release 0.3 [ exome aggregation consortium ; cambridge , ma ; http://exac.broadinstitute.org ] or > 10 healthy persons in the 1000 genomes project ) and a dominant inheritance model ( the allele was present in > 20 individuals in the 1000 genomes project or > 100 individuals from exac ) . segregation of the mutation with the phenotype in the family was verified by pcr and sanger sequencing using the bigdye protocol ( life technologies , carlsbad , ca ) on an abi 3500 genetic analyzer using following the oligonucleotides : forward : 5-tcc ttc aaa agg gca gtg tt-3 ; reverse : 5-cac cac tat ttc taa aca cgt ctc a-3. fragment length was analyzed with forward : 5-tgc tgt ctc cct aca caa aag-3 and reverse : fam - gaa aga atg aaa gac atg-3 oligonucleotides . ( e ) hematoxylin and eosin staining of the quadriceps muscle rules out grouped fiber atrophy . ( f ) staining with anti - myosin heavy chain ( fast ) antibodies reveals fiber type grouping as a sign of denervation / reinnervation . ( g ) electron microscopy of the neuromuscular endplate reveals condensation of the axoplasm and degeneration of axonal organelles ( open arrowhead ) , including mitochondria and loss of presynaptic neurofilaments , while the subneural clefts appear normal . five of 5 endplates that could be discovered in the muscle biopsy specimen had identical abnormalities . ( h ) normal neuromuscular endplate of the quadriceps muscle of an age - matched control at the same magnification for comparison . ( c ) genescan analysis of the insertion / deletion mutation at the intron 4 splice donor site , with homozygosity for the mutation in the patient and heterozygosity in all other family members . ( e ) investigation of receptor expression - enhancing protein 1 ( reep1 ) on the complementary dna from cultured fibroblasts verifies skipping of the 121 bp exon 4 . no normally spliced transcript was detected in the patient ; the mutant bands are faint , possibly because of nonsense - mediated messenger decay . co = control ; mut = mutant band ; ntc = nontemplate control ; wt = wild - type band . index patient and 2 control fibroblast lines were grown to confluency and trypsinized , and total rna was isolated using the rneasy kit ( qiagen , venlo , the netherlands ) reagent . total rna was reversely transcribed with superscript iii reverse transcriptase ( invitrogen , carlsbad , ca ) and random hexamers . oligonucleotide complementary dna primers flanking exon 4 ( forward : 5-cct gcg tat tat tcc tac aag gc-3 ; reverse : 5-ctt gga agc agc cat cac ag-3 ) were used for pcr , and the resulting products were separated by agarose electrophoresis and subjected to bidirectional automatic sanger sequencing . processing and staining of muscle cryostat and of transmission electron microscopic samples were performed as described previously . the study was approved by the institutional review board of the charit ( ea1/222/08 ) . parents provided written informed consent for all aspects of the study according to the declaration of helsinki . we performed initial autozygosity mapping followed by whole - exome sequencing ( wes ) of a consanguineous family with 1 affected patient ( figure 1 ) and 2 healthy siblings . for autozygosity mapping , we investigated the index patient ( patient ii:03 , figure 2a ) and 2 healthy siblings ( ii:01 and ii:02 ) using the affymetrix 6.0 genechip snp array and performed subsequent analysis using homozygositymapper2012 software ( http://www.homozygositymapper.org ) to identify genomic regions that were homozygous solely in the index patient . subsequent wes was performed on the index patient after capture with the sureselect v4 kit ( agilent technologies , santa clara , ca ) and sequencing on a hiseq 2000 machine ( illumina , san diego , ca ) , yielding 82 mio 2  101 bp paired - end reads that were aligned to the human grch37.p11 ( hg19/ensembl 72 ) genomic sequence . the mean coverage was 112 , and 87.9% of the captured sequences were covered more than 10 . after fine adjustment , the raw alignments were called for deviations from the reference in all coding exons 20 bp flanking regions . each of these variants was submitted to mutationtaster2 software to search for potentially pathogenic variants . these variants were further filtered for a recessive inheritance model ( the variant allele occurs in homozygous state in > 20 healthy individuals from the exac database release 0.3 [ exome aggregation consortium ; cambridge , ma ; http://exac.broadinstitute.org ] or > 10 healthy persons in the 1000 genomes project ) and a dominant inheritance model ( the allele was present in > 20 individuals in the 1000 genomes project or > 100 individuals from exac ) . segregation of the mutation with the phenotype in the family was verified by pcr and sanger sequencing using the bigdye protocol ( life technologies , carlsbad , ca ) on an abi 3500 genetic analyzer using following the oligonucleotides : forward : 5-tcc ttc aaa agg gca gtg tt-3 ; reverse : 5-cac cac tat ttc taa aca cgt ctc a-3. fragment length was analyzed with forward : 5-tgc tgt ctc cct aca caa aag-3 and reverse : fam - gaa aga atg aaa gac atg-3 oligonucleotides . ( e ) hematoxylin and eosin staining of the quadriceps muscle rules out grouped fiber atrophy . ( f ) staining with anti - myosin heavy chain ( fast ) antibodies reveals fiber type grouping as a sign of denervation / reinnervation . ( g ) electron microscopy of the neuromuscular endplate reveals condensation of the axoplasm and degeneration of axonal organelles ( open arrowhead ) , including mitochondria and loss of presynaptic neurofilaments , while the subneural clefts appear normal . five of 5 endplates that could be discovered in the muscle biopsy specimen had identical abnormalities . ( h ) normal neuromuscular endplate of the quadriceps muscle of an age - matched control at the same magnification for comparison . ( c ) genescan analysis of the insertion / deletion mutation at the intron 4 splice donor site , with homozygosity for the mutation in the patient and heterozygosity in all other family members . ( e ) investigation of receptor expression - enhancing protein 1 ( reep1 ) on the complementary dna from cultured fibroblasts verifies skipping of the 121 bp exon 4 . no normally spliced transcript was detected in the patient ; the mutant bands are faint , possibly because of nonsense - mediated messenger decay . co = control ; mut = mutant band ; ntc = nontemplate control ; wt = wild - type band . index patient and 2 control fibroblast lines were grown to confluency and trypsinized , and total rna was isolated using the rneasy kit ( qiagen , venlo , the netherlands ) reagent . total rna was reversely transcribed with superscript iii reverse transcriptase ( invitrogen , carlsbad , ca ) and random hexamers . oligonucleotide complementary dna primers flanking exon 4 ( forward : 5-cct gcg tat tat tcc tac aag gc-3 ; reverse : 5-ctt gga agc agc cat cac ag-3 ) were used for pcr , and the resulting products were separated by agarose electrophoresis and subjected to bidirectional automatic sanger sequencing . processing and staining of muscle cryostat and of transmission electron microscopic samples were performed as described previously . the now 5-year - old boy was born at term after a normal pregnancy to healthy first - cousin parents from lebanon . the patient presented with an equinovarus foot deformity , contractures of the distal phalanges , and a high - arched palate ( figure 1 , a  c ) . later , progressive distally pronounced weakness and hypotonia , mainly of the lower extremities , became evident . neurography of the sensory portion of the median nerve revealed axonal neuropathy with reduced amplitudes of 2.9 v ( normal 12.0  5.9 v ) but normal nerve conduction velocity of 43.1 m / s ( normal > 39.7 m / s ) . we were unable to elicit any measurable motor response or f - waves after stimulation of the median , peroneal , and tibial nerves . in contrast , muscle tendon reflexes were very brisk with enlarged trigger zones ( albeit without upgoing plantar reflexes ) , indicating upper motor neuron involvement ( video 1 at neurology.org/ng ) . mutation analysis of pmp22 , mpz , gdap1 , egr2 , sh3tc2 , ighmbp2 , and trpv4 genes had yielded normal results . electron microscopy of the axons and endplates ( figure 1 , g and h ) demonstrated loss of presynaptic neurofilaments and a condensed axoplasm with degeneration of organelles in the  bouton terminal . respiratory distress and paradoxical breathing due to diaphragmatic palsy were diagnosed at 11 months ( figure 1d , video 2 ) . following recurrent severe respiratory tract infections with acute respiratory failure , , the muscle weakness has not progressed ; however , he still can not sit without support . on the other hand , mental and speech development the healthy parents received a thorough clinical neurologic investigation and displayed normal muscle tone , strength , and reflexes as well as normal sensation , fine motor skills , and gait pattern . the only subtle abnormalities were a high palate and a mild increase in the foot arch of the father . neurography of motor ( median , tibial , peroneal ) and sensory ( median , sural ) nerves in both parents showed normal results . autozygosity mapping revealed several regions on chromosomes 1 , 2 , 10 , 11 , and 13 ( = 27.3 mbp ) , comprising 132 protein - coding genes . wes revealed 3 homozygous variants within the autozygous regions that were predicted to be potentially disease causing by mutationtaster2 software . the remaining homozygous splice site mutation in reep1 at the donor splice site of exon 4 ( c.303 + 1 - 7gtaatat > ac , p.f62kfs23 * ; nm_022912 ) was predicted to abrogate the splice site , resulting in a frameshift and premature termination codon after 23 nonoriginal amino acids . this variant was not listed in the exac database or the 1,000 genomes project as a known polymorphism . homozygosity of the mutation was confirmed by sanger sequencing and cosegregated with the phenotype in the family ( figure 2c ) . the splicing defect was verified on the level of the messenger rna ( mrna ) from fibroblasts and muscle ( figure 2e ) . repeated western blots ( anti - reep1 , sigma sab2101976 , 1:501:500 ) failed to show the presence of the reep1 protein in extracts from fibroblasts and muscle of the patient and of normal controls , possibly because of low expression levels in these tissues . finally , we performed molecular genetic testing of reep1 by sequencing all exons in 3 patients from a cohort of patients with a genetically unresolved smard1-like phenotype featuring clinical signs of additional involvement of the upper motor neuron . the now 5-year - old boy was born at term after a normal pregnancy to healthy first - cousin parents from lebanon . the patient presented with an equinovarus foot deformity , contractures of the distal phalanges , and a high - arched palate ( figure 1 , a  c ) . later , progressive distally pronounced weakness and hypotonia , mainly of the lower extremities , became evident . neurography of the sensory portion of the median nerve revealed axonal neuropathy with reduced amplitudes of 2.9 v ( normal 12.0  5.9 v ) but normal nerve conduction velocity of 43.1 m / s ( normal > 39.7 m / s ) . we were unable to elicit any measurable motor response or f - waves after stimulation of the median , peroneal , and tibial nerves . in contrast , muscle tendon reflexes were very brisk with enlarged trigger zones ( albeit without upgoing plantar reflexes ) , indicating upper motor neuron involvement ( video 1 at neurology.org/ng ) . mutation analysis of pmp22 , mpz , gdap1 , egr2 , sh3tc2 , ighmbp2 , and trpv4 genes had yielded normal results . electron microscopy of the axons and endplates ( figure 1 , g and h ) demonstrated loss of presynaptic neurofilaments and a condensed axoplasm with degeneration of organelles in the  bouton terminal . respiratory distress and paradoxical breathing due to diaphragmatic palsy were diagnosed at 11 months ( figure 1d , video 2 ) . following recurrent severe respiratory tract infections with acute respiratory failure , , the muscle weakness has not progressed ; however , he still can not sit without support . on the other hand , mental and speech development the healthy parents received a thorough clinical neurologic investigation and displayed normal muscle tone , strength , and reflexes as well as normal sensation , fine motor skills , and gait pattern . the only subtle abnormalities were a high palate and a mild increase in the foot arch of the father . neurography of motor ( median , tibial , peroneal ) and sensory ( median , sural ) nerves in both parents showed normal results . autozygosity mapping revealed several regions on chromosomes 1 , 2 , 10 , 11 , and 13 ( = 27.3 mbp ) , comprising 132 protein - coding genes . wes revealed 3 homozygous variants within the autozygous regions that were predicted to be potentially disease causing by mutationtaster2 software . the remaining homozygous splice site mutation in reep1 at the donor splice site of exon 4 ( c.303 + 1 - 7gtaatat > ac , p.f62kfs23 * ; nm_022912 ) was predicted to abrogate the splice site , resulting in a frameshift and premature termination codon after 23 nonoriginal amino acids . this variant was not listed in the exac database or the 1,000 genomes project as a known polymorphism . homozygosity of the mutation was confirmed by sanger sequencing and cosegregated with the phenotype in the family ( figure 2c ) . the splicing defect was verified on the level of the messenger rna ( mrna ) from fibroblasts and muscle ( figure 2e ) . no full - length mrna transcript was detected in the patient . repeated western blots ( anti - reep1 , sigma sab2101976 , 1:501:500 ) failed to show the presence of the reep1 protein in extracts from fibroblasts and muscle of the patient and of normal controls , possibly because of low expression levels in these tissues . finally , we performed molecular genetic testing of reep1 by sequencing all exons in 3 patients from a cohort of patients with a genetically unresolved smard1-like phenotype featuring clinical signs of additional involvement of the upper motor neuron . the known heterozygous dominant mutations in reep1 are associated with a slowly progressive form of hsp and are only rarely associated with a neuropathy . only one family with isolated motor axonal neuropathy without any signs of spasticity has been described . based on functional studies , the authors suspected that heterozygous loss - of - function mutations may result in upper motor neuron pathology , while mutations with a presumed toxic gain of function might cause lower motor neuron disease . the > 40 published dominant mutations of reep1 are evenly distributed over the entire reading frame , and neither clear genotype  phenotype correlation nor mutational hot spot has been established ( figure e-1 ) ; recessive mutations have not been reported . hence the location of the mutation in our family can not be linked to a special phenotype . of note , a previously reported mutation ( c.303g > a ) of the same exon 4 splice donor was associated with a pure hsp phenotype . in a reep1-deficient mouse model , animals developed a severe gait disorder resembling human hsp that was much more pronounced in homozygous animals than in heterozygous animals . in our patient , the functional relevance of the splice site mutation could be demonstrated on the mrna level in fibroblasts and muscle tissue but not on the protein level , presumably because reep1 expression levels are too low in these tissues to be detectable by western blot . these findings may hint at additional factors in the genetic background of the parents with an influence on reep1-related pathogenicity , although this remains speculative without having investigated further families with a similar phenotype . a similar constellation was reported for another disease associated with the dominant p.asn88ser berardinelli - seip congenital lipodystrophy type 2 gene ( bscl2 ) mutation , which had been detected in different unrelated families . this mutation is associated with a broad spectrum of neuromuscular disease phenotypes , ranging from motor neuropathy to complex hsp , but was also seen in asymptomatic heterozygous carriers . of note , homozygous bscl2 mutations are known to be associated with an entirely different disorder , congenital generalized lipodystrophy type 2 . similar to the bscl2 protein , reep1 is localized at the er and is relevant for er shaping . recently , it was shown in a drosophila model that overexpression of reep1 prevents tau - mediated neurodegeneration and promotes neuronal resistance to er stress . thus , reep1-associated disorders may also be summarized as er stress diseases , displaying different phenotypes depending on the type of disturbed er function . however , the precise underlying mechanisms remain to be elucidated . to date , only a few genes are known to be associated with a smard - like phenotype . both ighmbp2 ( smard1 ) and las1l ( smard2 with x - chromosomal inheritance ) play a role in rna processing , protein translation , and ribosomal biogenesis . in this respect , a function at the er might hint at a pathogenetic link between ighmbp2 and reep1 ; however , this requires further experimental evidence . in addition , mutations in the riboflavin transporters slc25a2 and slc2a3 may also mimic smard1 , just like mutations in megf10 , a gene involved in the regulation of satellite cells . our study expands the known phenotypical spectrum of reep1-associated neuromuscular disorders and highlights that recessive reep1 mutations should be considered in the genetic workup of patients with a smard1-like phenotype , especially if presenting with signs of upper motor neuron involvement . gudrun schottmann : recruited , investigated , and phenotyped the patients ; performed neurophysiologic investigations ; validated the results of the wes ; wrote the first draft of the manuscript . markus schuelke : investigated and phenotyped the patients ; performed the autozygosity mapping and haplotype analysis ; validated the results of the wes ; supervised the work and obtained funding support ; wrote the first draft of the manuscript . the project was funded by the deutsche forschungsgemeinschaft ( sfb 665 tp c4 ) to m.s . and the neurocure center of excellence ( exc 257 ) to m.s . gudrun schottmann , dominik seelow , franziska seifert , susanne morales - gonzalez , esther gill , katja von au , and arpad von moers report no disclosures . werner stenzel has served on the editorial boards of neuromuscular disorders and neuropathology and applied neurobiology .
objective : to identify the underlying genetic cause of a congenital neuropathy in a 5-year - old boy as part of a cohort of 32 patients from 23 families with genetically unresolved neuropathies.methods:we used autozygosity mapping coupled with next - generation sequencing to investigate a consanguineous family from lebanon with 1 affected and 2 healthy children . 
 variants were investigated for segregation in the family by sanger sequencing . 
 a splice site mutation was further evaluated on the messenger rna level by quantitative reverse transcription pcr . 
 subsequently , a larger cohort was specifically screened for receptor expression - enhancing protein 1 ( reep1 ) gene mutations.results:we detected a homozygous splice donor mutation in reep1 ( c.303 + 1 - 7gtaatat > ac , p.f62kfs23 * ; nm_022912 ) that cosegregated with the phenotype in the family , leading to complete skipping of exon 4 and a premature stop codon . 
 the phenotype of the patient is similar to spinal muscular atrophy with respiratory distress type 1 ( smard1 ) with additional distal arthrogryposis and involvement of the upper motor neuron manifested by pronounced hyperreflexia.conclusion:to date , only dominant reep1 mutations have been reported to be associated with a slowly progressive hereditary spastic paraplegia . 
 the findings from our patient expand the phenotypical spectrum and the mode of inheritance of reep1-associated disorders . 
 recessive mutations in reep1 should be considered in the molecular genetic workup of patients with a neuromuscular disorder resembling smard1 , especially if additional signs of upper motor neuron involvement and distal arthrogryposis are present .