the study protocol was approved by the local ethics committee , and written informed consent was obtained from the parents or guardians of each patient . we studied 44 probands with pndm diagnosed before 6 months of age , who had been referred to the molecular genetics laboratory at the peninsula medical school in exeter , u.k .. mutations in kcnj11 , abcc8 , ins , and gck had been excluded . the relevant clinical information was obtained from the medical records . the single coding exon of neurod1 was pcr amplified in three overlapping fragments using specific primers for each amplicon tagged with 5 m13 tails to allow sequencing to be performed with a universal m13 primer ( primers and conditions available upon request ) . single - strand sequencing was carried out using standard methods on an abi 3730 sequencer ( applied biosystems , warrington , u.k . ) . high - density single nucleotide polymorphism ( snp ) genotyping was carried out on the affymetrix human 10k xba chip by medical solutions nottingham ( formerly geneservice ) ( nottingham , u.k . ) . house perl scripts were developed to automatically identify genomic homozygous segments , defined by at least 20 consecutive homozygous snps marking a region that exceeded 3 cm ( 13 ) . we studied 44 probands with pndm diagnosed before 6 months of age , who had been referred to the molecular genetics laboratory at the peninsula medical school in exeter , u.k .. mutations in kcnj11 , abcc8 , ins , and gck had been excluded . the single coding exon of neurod1 was pcr amplified in three overlapping fragments using specific primers for each amplicon tagged with 5 m13 tails to allow sequencing to be performed with a universal m13 primer ( primers and conditions available upon request ) . single - strand sequencing was carried out using standard methods on an abi 3730 sequencer ( applied biosystems , warrington , u.k . ) . high - density single nucleotide polymorphism ( snp ) genotyping was carried out on the affymetrix human 10k xba chip by medical solutions nottingham ( formerly geneservice ) ( nottingham , u.k . ) . processing of genomic dna was performed in accordance with the affymetrix protocol . in - house perl scripts were developed to automatically identify genomic homozygous segments , defined by at least 20 consecutive homozygous snps marking a region that exceeded 3 cm ( 13 ) . two novel homozygous mutations in neurod1 , a single base pair duplication ( c.364dupg ) and a two - base pair ct deletion ( c.427_428del ) , were identified in two unrelated probands . both mutations result in a frameshift and a premature truncation of the c terminus of the expressed protein ( p.asp122glyfs*12 and p.leu143alafs*55 , respectively ) , leading to mutated proteins completely lacking the transactivation domain ( fig . these mutations had not been previously documented and were not present in 200 alleles from healthy unrelated individuals . the abnormal protein sequence between the frameshift and the termination codon is colored in gray . a with the c.364dupg mutation , parents were known to be first cousins and , consistent with parental consanguinity , snp genotyping analysis of the proband revealed a total genomic homozygosity value of 6.0% ( 13 ) . the mutation - containing homozygous segment was the largest homozygous segment ( 46.6-mb long ) and spanned 2q31.12q36.1 delimited by the snps rs726032 to rs724149 . in contrast , in family b , the parents of the patient with the homozygous c.427_428del mutation were not known to be related and , in keeping with this , total genomic homozygosity value was very low ( 0.3% ) . however , the mutation in both parents was inherited on an extended haplotype of 10.4 mb between positions chr2q31.132.1 ( snps rs2884471rs722385 ) , suggesting that the mutation arose from a single common ancestor . extended pedigrees of the two families showing inheritance of neurod1 mutations ( family a , c.364dupg [ a ] ; family b , c427_428del [ b ] ) . genotype is shown underneath each symbol ; m and n denote mutant and wild - type alleles , respectively . directly below the genotype is the age of the individual at testing or the age at diagnosis of diabetes if diabetic , followed by the most recent treatment for diabetes . black - filled symbols denote patients with neonatal diabetes , and gray - filled symbols represent patients with later - onset diabetes . the two probands were diagnosed with permanent diabetes within the first 2 months of life and had presented with intrauterine growth retardation ( birth weights 1,490 and 2,230 g at 34 and 38 weeks of gestation , respectively ) , reflecting reduced insulin secretion in utero . they had no evidence of pancreatic exocrine dysfunction and normal pancreatic size on abdominal scanning ( see supplementary information , available in an online appendix [ http://diabetes.diabetesjournals.org/cgi/content/full/db10-0011/dc1 ] ) . in addition to diabetes , they presented with a similar pattern of neurological abnormalities including moderate - to - severe developmental delay , profound sensorineural deafness , and visual impairment due to myopia and diffuse retinal dystrophy . brain magnetic resonance imaging scans showed severe cerebellar hypoplasia with no other major intracranial abnormalities ( fig . 3 and supplementary information ) . magnetic resonance imaging of the brain in a proband from family a demonstrating the typical neuroimaging findings of neurod1-pndm . a : sagittal t1-weighted image . b : coronal t2-weighted image . there is significant cerebellar hypoplasia , particularly of cerebellar vermis inferiorly . . clinical features of the two patients with homozygous neurod1 mutations ct , computed tomography ; erg , electroretinography ; mri , magnetic resonance imaging ; sds , sd score the diabetes status , age of diagnosis , treatment , and genetic testing result of family members are shown in fig . 2 . we assessed glucose tolerance in the four parents of the two probands who were proven heterozygous carriers of the mutations . in family a ( c.364dupg mutation ) , the mother had been diagnosed with type 2 diabetes at 33 years , despite having a normal bmi , and was treated with glicazide . in contrast , the father ( also aged 33 years ) had normal fasting ( 46 mmol / l ) and postprandial ( 57 mmol / l ) blood glucose levels on several occasions . in family b ( c.427_428del mutation ) , the mother and father underwent standard oral glucose tolerance tests ( aged 33 and 37 years , respectively ) that confirmed normal glucose tolerance ( 6.2 and 4.8 mmol / l , respectively , at 2 h ) . no heterozygous family members in either family had any developmental delay or neurological features on clinical examination . two novel homozygous mutations in neurod1 , a single base pair duplication ( c.364dupg ) and a two - base pair ct deletion ( c.427_428del ) , were identified in two unrelated probands . both mutations result in a frameshift and a premature truncation of the c terminus of the expressed protein ( p.asp122glyfs*12 and p.leu143alafs*55 , respectively ) , leading to mutated proteins completely lacking the transactivation domain ( fig . these mutations had not been previously documented and were not present in 200 alleles from healthy unrelated individuals . the abnormal protein sequence between the frameshift and the termination codon is colored in gray . a with the c.364dupg mutation , parents were known to be first cousins and , consistent with parental consanguinity , snp genotyping analysis of the proband revealed a total genomic homozygosity value of 6.0% ( 13 ) . the mutation - containing homozygous segment was the largest homozygous segment ( 46.6-mb long ) and spanned 2q31.12q36.1 delimited by the snps rs726032 to rs724149 . in contrast , in family b , the parents of the patient with the homozygous c.427_428del mutation were not known to be related and , in keeping with this , total genomic homozygosity value was very low ( 0.3% ) . however , the mutation in both parents was inherited on an extended haplotype of 10.4 mb between positions chr2q31.132.1 ( snps rs2884471rs722385 ) , suggesting that the mutation arose from a single common ancestor . extended pedigrees of the two families showing inheritance of neurod1 mutations ( family a , c.364dupg [ a ] ; family b , c427_428del [ b ] ) . genotype is shown underneath each symbol ; m and n denote mutant and wild - type alleles , respectively . directly below the genotype is the age of the individual at testing or the age at diagnosis of diabetes if diabetic , followed by the most recent treatment for diabetes . black - filled symbols denote patients with neonatal diabetes , and gray - filled symbols represent patients with later - onset diabetes . the two probands were diagnosed with permanent diabetes within the first 2 months of life and had presented with intrauterine growth retardation ( birth weights 1,490 and 2,230 g at 34 and 38 weeks of gestation , respectively ) , reflecting reduced insulin secretion in utero . they had no evidence of pancreatic exocrine dysfunction and normal pancreatic size on abdominal scanning ( see supplementary information , available in an online appendix [ http://diabetes.diabetesjournals.org/cgi/content/full/db10-0011/dc1 ] ) . in addition to diabetes , they presented with a similar pattern of neurological abnormalities including moderate - to - severe developmental delay , profound sensorineural deafness , and visual impairment due to myopia and diffuse retinal dystrophy . brain magnetic resonance imaging scans showed severe cerebellar hypoplasia with no other major intracranial abnormalities ( fig . 3 and supplementary information ) . magnetic resonance imaging of the brain in a proband from family a demonstrating the typical neuroimaging findings of neurod1-pndm . a : sagittal t1-weighted image . b : coronal t2-weighted image . there is significant cerebellar hypoplasia , particularly of cerebellar vermis inferiorly . clinical features of the two patients with homozygous neurod1 mutations ct , computed tomography ; erg , electroretinography ; mri , magnetic resonance imaging ; sds , sd score . the diabetes status , age of diagnosis , treatment , and genetic testing result of family members are shown in fig . 2 . we assessed glucose tolerance in the four parents of the two probands who were proven heterozygous carriers of the mutations . in family a ( c.364dupg mutation ) , the mother had been diagnosed with type 2 diabetes at 33 years , despite having a normal bmi , and was treated with glicazide . in contrast , the father ( also aged 33 years ) had normal fasting ( 46 mmol / l ) and postprandial ( 57 mmol / l ) blood glucose levels on several occasions . in family b ( c.427_428del mutation ) , the mother and father underwent standard oral glucose tolerance tests ( aged 33 and 37 years , respectively ) that confirmed normal glucose tolerance ( 6.2 and 4.8 mmol / l , respectively , at 2 h ) . no heterozygous family members in either family had any developmental delay or neurological features on clinical examination . the patients with this novel autosomal recessive syndrome not only had early - onset permanent diabetes but also presented with developmental delay , cerebellar hypoplasia , and hearing and visual impairment . this is the 13th gene in which mutations have been described in patients with permanent neonatal diabetes . neurod1 , a tissue - specific member of the bhlh family of transcription factors , is expressed in developing pancreatic islets and in mature -cells . it forms a heterodimer with the ubiquitous bhlh transcription factor e47 that binds to specific e - box motifs on specific target genes , including ins , gck , and abcc8 , to regulate their expression ( 1416 ) . the two homozygous neurod1 mutations both introduce a frameshift that results in truncated proteins lacking the transactivation domain , which has been shown to be important for the interaction of neurod1 with its main coactivator , p300 ( 17 ) . these are likely to have no biological activity , as shown previously for a different frameshift mutation ( c.616dupc , p.his206profs*38 ) identified in a patient with neurod1-mody ( 9 ) . the two patients have a remarkably consistent phenotype ( table 1 ) , with clinical features in keeping with the known expression and biology of this transcription factor , and this provides further evidence that the homozygous mutations in neurod1 are causative . both patients have neonatal diabetes but a normal pancreas on scanning and no evidence of exocrine dysfunction . histological examination of the neurod1-deficient pancreas shows an impaired islet morphogenesis with a reduction in the number of endocrine cells  especially -cells ( 8) . in addition to diabetes , our two patients presented with a similar pattern of neurological features , including developmental delay , cerebellar hypoplasia , and visual and hearing impairment . this is in keeping with the abundant expression of neurod1 in the developing and mature nervous system . interestingly , the initial neurod1-null mice that rapidly died from diabetes had no obvious anatomic and histologic abnormalities of the brain ( 8) . however , it is possible to explore the role of neurod1 in the nervous system by rescuing neurod1-null mice either by expressing a transgene encoding the mouse neurod1 gene under the insulin promoter ( 18 ) or by crossing the null mutation into a different genetic background to reduce the severity of the diabetes ( 19 ) . the rescued neurod1-null mice show a similar neuronal phenotype consisting of impaired balance , ataxic gait , circling , and swaying head movement as a result of impaired cerebellum development ( 1820 ) . furthermore , rescued neurod1-deficient mice have abnormal hearing and vision as a result of severe sensory neuronal defects in the inner ear and neural retina , respectively ( 2022 ) . the main feature seen in the mouse that was not present in our patients was epilepsy ( 19 ) . the remarkable similarity between the neurod1-deficient patients and the neurod1-deficient mice ( table 2 ) strongly supports a similar biological role of this transcription factor across species . comparison of the major features seen in neurod1-deficient mice ( refs . 8 and 1822 ) and neurod1-deficient patients with homozygous neurod1 mutations early - onset ketosis - prone diabetes failure of mature islets development striking reduction in both beta and alpha cells permanent neonatal diabetes postnatal - onset acinar cell polarity defects ( indirect effect ? ) lack of secretin- and cholecystokinin - producing cells ( remaining enteroendocrine cells normal ) > 95% decrease of granule cells impaired coordination and ataxia decrease of granule cells decreased synapses and loss of outer nuclear layer deafness and imbalance shortened cochlear duct , sensory epithelia abnormalities , and degeneration of acoustic ganglions sensorineural deafness homozygous mutations in ptf1a , which encodes another bhlh transcription factor , also cause a syndrome of neonatal diabetes and cerebellar hypoplasia / agenesis ( 23 ) . however , in this condition the pancreatic phenotype is not limited to the islets ; affected patients have pancreatic hypoplasia / aplasia . in keeping with the islets representing < 1% of the endocrine pancreas , the size of the pancreas was found to be normal in our two patients with homozygous neurod1 mutations . this suggests that shared developmental pathways are important during development in the pancreas and the cerebellum . although heterozygous loss - of - function mutations in neurod1 have previously been identified as a very rare cause of diabetes in humans ( 912 ) , diabetes was present in only one of four heterozygous mutation  carrying parents . their age at the time of the study ranged from 33 to 39 years and does not exclude the possibility of developing diabetes later in life . in addition , incomplete penetrance has been described in some of the families with neurod1 diabetes ( 9 ) . homozygous mutations in other known mody genes , namely gck and ipf1 , have previously been associated with isolated pndm and isolated pancreatic agenesis , respectively ( 24,25 ) . we have shown that homozygous mutations in another mody gene are also associated with a more severe phenotype of neonatal diabetes . in conclusion , homozygous mutations in neurod1 constitute a rare novel autosomal recessive cause of neonatal diabetes with severe neurological abnormalities . this confirms the important role that neurod1 plays in the development of both the pancreas and the nervous system in humans .