X-linked progressive mixed deafness: a new microdeletion that involves a more proximal region in Xq21.

We report a large two-generation pedigree with seven affected males segregating for an X-linked mixed conductive sensorineural deafness. The patients present with atypical Mondini-like dysplasia, dilated petrous facial canal, dilatation of the internal auditory meatus fully connected with enlarged cochlear canals, and, in one patient, a wide bulbous posterior labyrinth. Obligatory carrier females are mildly affected. Molecular characterization of this family revealed a deletion of locus DXS169, in Xq21.1. Loci DXS72 and DXS26, which, respectively, flank DXS169 proximally and distally, were intact. Since a gene responsible for X-linked progressive mixed deafness with perilymphatic gusher (DFN3) has previously been assigned by deletion mapping to a slightly more distal interval between DXS26 and DXS121, this study indicates either two different deafness genes or the involvement of a very large region in Xq21.     

A novel autosomal recessive non-syndromic deafness locus, DFNB66, maps to chromosome 6p21.2-22.3 in a large Tunisian consanguineous family

Hereditary non-syndromic deafness is extremely heterogeneous. Autosomal recessive forms account for approximately 80% of genetic cases. Autosomal recessive non-syndromic sensorineural deafness segregating in a large consanguineous Tunisian family was mapped to chromosome 6p21.2-22.3. A maximum lod score of 5.36 at theta=0 was obtained for the polymorphic microsatellite marker IR2/IR4. Haplotype analysis defined a 16.5-Mb critical region between microsatellite markers D6S1602 and D6S1665. The screening of 3 candidate genes, COL11A2, BAK1 and TMHS, did not reveal any disease causing mutation, suggesting that this is a novel deafness locus, which has been named DFNB66. A search in the Human Cochlear EST Library for ESTs located in this critical interval allowed us to identify several candidates. Further investigations on these candidates are needed in order to identify the deafness-causing gene in this Tunisian family.     

A unique point mutation in the PMP22 gene is associated with Charcot-Marie-Tooth disease and deafness.

Charcot-Marie-Tooth disease (CMT) with deafness is clinically distinct among the genetically heterogeneous group of CMT disorders. Molecular studies in a large family with autosomal dominant CMT and deafness have not been reported. The present molecular study involves a family with progressive features of CMT and deafness, originally reported by Kousseff et al. Genetic analysis of 70 individuals (31 affected, 28 unaffected, and 11 spouses) revealed linkage to markers on chromosome 17p11.2-p12, with a maximum LOD score of 9.01 for marker D17S1357 at a recombination fraction of .03. Haplotype analysis placed the CMT-deafness locus between markers D17S839 and D17S122, a approximately 0.6-Mb interval. This critical region lies within the CMT type 1A duplication region and excludes MYO15, a gene coding an unconventional myosin that causes a form of autosomal recessive deafness called DFNB3. Affected individuals from this family do not have the common 1.5-Mb duplication of CMT type 1A. Direct sequencing of the candidate peripheral myelin protein 22 (PMP22) gene detected a unique G-->C transversion in the heterozygous state in all affected individuals, at position 248 in coding exon 3, predicted to result in an Ala67Pro substitution in the second transmembrane domain of PMP22.     

Temperature-sensitive auditory neuropathy associated with an otoferlin mutation: Deafening fever!

Transient deafness associated with an increase in core body temperature is a rare and puzzling disorder. Temperature-dependent deafness has been previously observed in patients suffering from auditory neuropathy. Auditory neuropathy is a clinical entity of sensorineural deafness characterized by absent auditory brainstem response and normal otoacoustic emissions. Mutations in OTOF, which encodes otoferlin, have been previously reported to cause DFNB9, a non-syndromic form of deafness characterized by severe to profound prelingual hearing impairment and auditory neuropathy.Here we report a novel mutation in OTOF gene in a large family affected by temperature-dependent auditory neuropathy. Three siblings aged 10, 9 and 7 years from a consanguineous family were found to be affected by severe or profound hearing impairment that was only present when they were febrile. The non-febrile patients had only mild if any hearing impairment. Electrophysiological tests revealed auditory neuropathy. Mapping with microsatellite markers revealed a compatible linkage in the DFNB9/OTOF region in the family, prompting us to run a molecular analysis of the 48 exons and of the OTOF intron-exon boundaries. This study revealed a novel mutation p.Glu1804del in exon 44 of OTOF. The mutation was found to be homozygous in the three patients and segregated with the hearing impairment within the family. The deletion affects an amino acid that is conserved in mammalian otoferlin sequences and located in the calcium-binding domain C2F of the protein.     

X-ray diagnosis of different forms of Hirschsprung's disease in children

The purpose of this study was to examine and compare X-ray signs and roentgenometric parameters in different forms of childhood Hirschsprung's disease for their more accurate diagnosis. A hundred and thirty-eight children with different forms of Hirschsprung's disease were followed up. Among them, the children with a supershort segment of the disease amounted to 55%. All the children underwent a comprehensive examination including barium irrigography according to the procedure described by M.D. Levin, followed by an estimation of roentgenometric parameters. The long forms of Hirschsprung's disease had characteristic X-ray symptoms and their appropriate roentgenometric parameters. The supershort segment of the disease had not a complete complex of characteristic X-ray symptoms and its roentgenometric parameters were in direct opposition to the similar parameters in the long form of this disease.     

A novel mitochondrial DNA missense mutation at G3421A in a family with maternally inherited diabetes and deafness

OBJECTIVE: Mutations in mtDNA are thought to be responsible for the pathogenesis of maternally inherited diabetes. Here, we report a family with maternally inherited diabetes and deafness whose members did not harbour the mtDNA A3243G mutation, the most frequent point mutation in mitochondrial diabetic patients. This study aimed to investigate a possible other mtDNA mutation and its prevalence in type 2 diabetic patients. METHODS: Height, body weight, waistline, and hip circumference were measured and serum biochemical marks determined in all members of the family. In addition, a 75 g oral glucose tolerance test and electric listening test were conducted in these members. Genomic DNA was prepared from peripheral leukocytes. Direct sequencing of PCR products was used to detect the mtDNA mutation in this family. The prevalence of mtDNA G3421A nucleotide substitutions was investigated by restriction fragment length polymorphism analysis in 1350 unrelated type 2 diabetic patients recruited by random cluster sampling from the central city area of Shanghai, China. RESULTS: (1) A new missense homoplasmic mutation of mtDNA G3421A was found in a maternally inherited diabetic family and existed neither in 1350 unrelated type 2 diabetic patients nor in 50 non-diabetic individuals. (2) The mode of mutation and diabetes transmission was typical maternal inheritance in this family. (3) All diabetic family members were found to have an onset at 35-42 years of age, accompanied by deafness of varying degrees. CONCLUSION: mtDNA G3421A (Val39Ile) found in a family with maternally inherited diabetes and deafness is a novel missense mutation. Whether this is a diabetogenic mutation and its effect on mitochondrial function needs to be further studied.     

Analysis of two Arab families reveals additional support for a DFNB2 nonsyndromic phenotype of MYO7A

Variants in the head and tail domains of the MYO7A gene, encoding myosin VIIA, cause Usher syndrome type 1B (USH1B) and nonsyndromic deafness (DFNB2, DFNA11). In order to identify the genetic defect(s) underling profound deafness in two consanguineous Arab families living in UAE, we have sequenced a panel of 19 genes involved in Usher syndrome and nonsyndromic deafness in the index cases of the two families. This analysis revealed a novel homozygous insertion of AG (c.1952_1953insAG/p.C652fsX11) in exon 17 of the MYO7A gene in an Iraqi family, and a homozygous point mutation (c.5660C>T/p.P1887L) in exon 41 affecting the same gene in a large Palestinian family. Moreover, some individuals from the Palestinian family also harbored a novel heterozygous truncating variant (c.1267C>T/p.R423X) in the DFNB31 gene, which is involved in autosomal recessive nonsyndromic deafness type DFNB31 and Usher syndrome type II. Assuming an autosomal recessive mode of inheritance in the two inbred families, we conclude that the homozygous variants in the MYO7A gene are the disease-causing mutations in these families. Furthermore, given the absence of retinal disease in all affected patients examined, particularly a 28 year old patient, suggests that at least one family may segregate a DFNB2 presentation rather than USH1B. This finding further supports the premise that the MYO7A gene is responsible for two distinct diseases and gives evidence that the p.P1887L mutation in a homozygous state may be responsible for nonsyndromic hearing loss.     

近亲结婚所致一遗传性非综合征型耳聋家系的调查

耳聋是一种最常见的人类感觉系统缺陷, 在已发现的遗传性耳聋中,有70％的属于非综合征型听力缺损。据估计非综合征型遗传性耳聋基因总数在100个以上,目前已经确定了近80个非综合征型遗传性耳聋的遗传位点,其中23个基因已经被成功克隆。文章报道一遗传性非综合征型耳聋家系。该家系中存在2代近亲结婚,共2代13人出现聋哑症状。经遗传分析,该家系的遗传方式与常染色体显性或隐性遗传均不符合,提示此家系中的非综合征型遗传性耳聋可能为线粒体突变所致。     

MtDNA mutations in maternally inherited diabetes: presence of the 3397 ND1 mutation previously associated with Alzheimer's and Parkinson's disease

Mutations in the mitochondrial tRNA(leu) (UUR) gene have been associated with diabetes mellitus and deafness. We screened for the presence of mtDNA mutations in the tRNA(leu) (UUR) gene and adjacent ND1 sequences in 12 diabetes mellitus pedigrees with a possible maternal inheritance of the disease. One patient carried a G to A substitution at nt 3243 (tRNA(leu) (UUR) gene) in heteroplasmic state. In a second pedigree a patient had an A to G substitution at nt 3397 in the ND1 gene. All maternal relatives of the proband had the 3397 substitution in homoplasmic state. This substitution was not present in 246 nonsymptomatic Caucasian controls. The 3397 substitution changes a highly conserved methionine to a valine at aa 31 and has previously been found in Alzheimer's (AD) and Parkinson's (PD) disease patients. Substitutions in the mitochondrial ND1 gene at aa 30 and 31 have associated with a number of different diseases (e.g. AD/PD, MELAS, cardiomyopathy and diabetes mellitus, LHON, Wolfram-syndrome and maternal inherited diabetes) suggesting that changes at these two codons may be associated with very diverse pathogenic processes. In a further attempt to search for mtDNA mutations outside the tRNAleu gene associated with diabetes, the whole mtDNA genome sequence was determined for two patients with maternally inherited diabetes and deafness. Except for substitutions previously reported as polymorphisms, none of the two patients showed any non-synonymous substitutions either in homoplasmic or heteroplasmic state. These results imply that the maternal inherited diabetes and deafness in these patients must result from alterations of nuclear genes and/or environmental factors.     

Distribution, quantitation, and origin of immunoreactive neuropeptide Y in the human gastrointestinal tract

A radioimmunoassay for measurement of immunoreactive neuropeptide Y has been developed using antiserum from a rabbit (221) immunized with porcine neuropeptide Y. Antibody 221 has been characterized for both sensitivity and specificity. To determine the distribution of neuropeptide Y in the human gastrointestinal tract, fresh tissue specimens were separated by microdissection into the muscularis externa and the mucosa-submucosa. To examine the origin of neuropeptide Y in human colon, specimens of aganglionic and ganglionic colon were obtained from patients with Hirschsprung's disease. Immunoreactive neuropeptide Y in human gut was present in highest concentrations in the muscularis externa of the stomach and in lowest concentrations in the muscularis externa of the ileum and descending colon. Neuropeptide Y in the stomach was present in higher concentrations in the muscularis externa than in the mucosa-submucosa, but in the descending colon there were lower concentrations of neuropeptide Y in the muscularis externa than in the mucosa-submucosa. In Hirschsprung's disease, concentrations of neuropeptide Y were increased in aganglionic colon in both the muscularis externa and the mucosa-submucosa, compared to corresponding layers from proximal ganglionic colon. Extracts of the gastric muscularis externa and the colonic mucosa-submucosa were separated by C18 reverse-phase high-performance liquid chromatography. One major immunoreactive species was identified by radioimmunoassay which eluted in a position similar to synthetic human neuropeptide Y. These results demonstrated both regional and layer differences in concentrations of neuropeptide Y in human gut. Increased concentrations of neuropeptide Y in aganglionic colon from Hirschsprung's disease most likely result from enlargement of neuropeptide Y-containing extrinsic nerve fibers in both the mucosa-submucosa and the muscularis externa.     

Targeted Next-Generation Sequencing in Uyghur Families with Non-Syndromic Sensorineural Hearing Loss

The mutation spectrum of deafness genes may vary in different ethnical groups. In this study, we investigated the genetic etiology of nonsyndromic deafness in four consanguineous and two multiplex Uyghur families in which mutations in common deafness genes GJB2, SLC26A4 and MT-RNR1 were excluded. Targeted next-generation sequencing of 97 deafness genes was performed in the probands of each family. Novel pathogenic mutations were identified in four probands including the p.L416R/p.A438T compound heterozygous mutations in TMC1, the homozygous p.V1880E mutation in MYO7A, c.1238delT frameshifting deletion in PCDH15 and c.9690+1G>A splice site mutation in MYO15A. Co-segregation of the mutations and the deafness were confirmed within each family by Sanger sequencing. No pathogenic mutations were identified in one multiplex family and one consanguineous family. Our study provided a useful piece of information for the genetic etiology of deafness in Uyghurs.     

Expression of leukaemia inhibitory factor during the development of the human enteric nervous system

Leukaemia inhibitory factor (LIF) is a neuropoietic cytokine, which promotes the development of enteric neurons in vitro, particularly when administered together with neurotrophin-3 (NT-3). The purpose of this study was to map the LIF immunoreactivity in the human enteric nervous system in foetuses, children, adults, and in patients with Hirschsprung's disease. Normal bowel specimens were obtained at postmortem examination of 13 foetuses, at 13–31 weeks of gestation, and at surgery in five children and two adults. Bowel resected in seven patients with Hirschsprung's disease was also investigated. Immunohistochemical analysis was performed on material fixed in formalin and embedded in paraffin. The specimens were exposed to antibodies raised against LIF. The ABC-complex method was used to visualise binding of antibodies to the corresponding antigen. LIF immunoreactivity was disclosed in the myenteric and submucous ganglion cells at 13–31 weeks of gestation, in childhood cases, and adults. LIF-immunoreactive ganglion cells were absent in aganglionic bowel, where the ganglia in the intermuscular layer were replaced by hypertrophic nerve bundles. These morphological findings indicate that LIF may play a role in the development of the enteric nervous system.     

先天性巨结肠病结肠壁内P物质能神经的免疫电镜观察

本研究应用电镜免疫细胞化学方法,对小儿先天性巨结肠病结肠壁内含Ｐ物质（ＳＰ）神经进行了观察。结果发现：狭窄段与结肠扩张段相比较,狭窄段含ＳＰ神经元缺失,神经终末减少,含无颗粒囊泡的神经终末增多,而含小颗粒囊泡的神经终末相对较少。本研究在超微结构水平为先天性巨结肠病结肠壁内肽能神经成份的改变及其与临床症状的可能关系提供了形态学证据。     

Change in the Distribution of Acetylcholinesterase Molecular Forms in Hirschsprung's Disease

Abstract: Density gradient ultracentrifugation shows that two molecular forms of acetylcholinesterase (4S and 10S) can be distinguished in the bowels of both normal subjects and Hirschsprung's disease patients. In this disease, besides the very large elevation of acetylcholinesterase activity, the relative distribution of the heavy and light forms was also changed. In the affected bowel the 10S/4S ratio was 2.5 times higher than the normal value. It is assumed that the accumulation of the 10S form might be a response of the intestine to this pathological state. It is also suggested that the increase in the heavy form is closely connected with the nerve fibre proliferation in the aganglionic megacolon.     

Segmental distribution of colonic neuropeptides in Hirschsprung's disease.

Despite continued research, the pathophysiologic mechanism responsible for functional obstruction in the aganglionic segment of bowel in Hirschsprung's disease remains controversial. Narrowing of the affected segment is thought by many investigators to be the result of loss of intrinsic inhibitory innervation. For this hypothesis to be consistent, inhibitory neuropeptides should be present in the dilating, transitional segment of bowel. In order to quantitate reported changes in peptidergic nerve staining in Hirschsprung's disease, we measured concentrations of five neuropeptides (vasoactive intestinal peptide, peptide histidine-methionine, met5-enkephalin, substance P and bombesin-like immunoreactivity) by radioimmunoassay in the affected segments of bowel from six patients with Hirschsprung's disease. Tissue extracts were prepared using gut obtained at surgery from the: (1) constricted, aganglionic segment, (2) dilating, aganglionic transitional segment and (3) dilated, proximal ganglionic segment. Concentrations of vasoactive intestinal peptide, peptide histidine-methionine, substance P and met5-enkephalin were significantly reduced in both the muscularis externa and the mucosal-submucosal layers from the constricted aganglionic segment. By contrast, concentrations of the candidate inhibitory neuropeptides, vasoactive intestinal peptide and peptide histidine-methionine, were minimally reduced in the dilating, aganglionic transitional segment. These results are consistent with the hypothesis that constriction of the aganglionic segment is due to loss of intrinsic inhibitory innervation. Concentrations of bombesin-like immunoreactivity were similar in the three segments of human gut, suggesting the presence of this immunoreactive neuropeptide in extrinsic nerve fibers.     

一个母系遗传非综合征耳聋大家系mtDNA序列分析

通过分析本家mtDNA序列,探讨淮阴一非综合耳聋大家患病的分子遗传学机制。采用聚合酶链反应（PCR）扩增mtDNA与非综合征耳聋相关位点nt1555,nt7445的区域和人类种群研究的D－loop区,PCR－异源双链分析,PCR－RFLP、PCR产物克隆序列测定等技术对该家系进行了系统的研究。发现该家系中全部母系亲属有mtDNAA1555G突变,而家系中非母 个体,对照组（100例正常个体）的mtDNA1555位点均为A。该家系mtDNA7445位点无突变;该系属于Ⅱ型线性体;发现家系D－loop区存在未见报道的碱基插入。提示mtDNAA1555G位点突变可能是导致该家系患致聋的主要因素之一。遗传背景可能对家系疾病的表现存在一定程度的影响。     

Smooth muscle from aganglionic bowel in Hirschsprung's disease impairs neuronal development in vitro

Hirschsprung's disease results from the congenital absence of enteric neurons in human distal colon. The reason for aganglionosis is unknown but may reflect an unfavourable microenvironment for neuronal development. We asked if smooth muscle cells from the aganglionic region could affect neuronal development in vitro. Neurons from neonatal mouse superior cervical ganglia were added to cultures of smooth muscle obtained from normal or aganglionic regions of five patients with Hirschsprung's disease. Although neurons initially showed more rapid attachment to aganglionic smooth muscle, this was equal by 60 min and thereafter. Progressive increase in the diameter of the nerve cell body was characteristic of normal maturation in vitro. This was consistently inhibited by 15–22% in neurons grown on aganglionic muscle compared with normal controls over the 6-day test period (P<0.05). This phenomenon was preserved when the smooth muscle cells were lysed by brief exposure to distilled water before initiation of coculture (16–18% inhibition; P<0.05). These data imply that smooth muscle of the aganglionic colon is less favourable for neuronal development than the normally innervated region and suggest a membrane-linked factor. Clearly, this persists in postnatal life and in vitro and may reflect an abnormality of cellular interaction causing Hirschsprung's disease.     

Genetic heterogeneity among kindreds with Alport syndrome.

Twenty-three kindreds were ascertained through patients at renal clinics at University of Utah Associated Hospitals. Urinalysis indicated glomerulonephritis in 231 of 997 examined kindred members; medical records documented kidney disease consistent with glomerulonephritis in 88 unexamined kindred members. Renal biopsies of 35 persons in a subset of 14 kindreds showed ultrastructural changes and absence of immune phenomena consistent with the diagnosis of Alport syndrome. End-stage renal disease (ESRD) had occurred in 72 (49%) of 148 known affected males and in 13 (8%) of 171 known affected females. No father-son affected pairs occurred in any of the kindreds; 84% of daughters of affected fathers were affected, and 49% of sons and 48% of daughters of affected mothers were affected. One of three phenotypes (juvenile Alport syndrome with deafness, adult Alport syndrome with deafness, or adult Alport syndrome without deafness or other defects) occurred in each of the 23 kindreds. We applied likelihood analysis to test for genetic heterogeneity underlying the phenotypic heterogeneity. In the first application (the admixture test), we tested for the occurrence of two forms of the disease without specifying which kindred had which form; we found insufficient evidence of admixture. In the second application (the predivided-sample test), we tested for genetic heterogeneity expressed as phenotypic heterogeneity. Kindreds were successively divided into two subgroups, with admission to the first subgroup dependent upon: (1) having greater than or equal to 2 males with ESRD, (2) occurrence of deafness in most nephrologically affected male family members, and (3) intrakindred mean age of ESRD in males later than age 31. Weak evidence of heterogeneity was found for category (1); stronger evidence of heterogeneity was found for category (3). Penetrance of microscopic hematuria in female heterozygotes was estimated as 82% overall, 85% for adult Alport syndrome, and 28% for juvenile Alport syndrome.     

Moderate hearing loss and pseudodominant inheritance due to L90P/35delG mutations in the GJB2 (connexin 26) gene

Mutations in the GJB2 (connexin 26-Cx26) gene are responsible for 20-50% of cases with prelingual non-syndromic deafness in a large part of the world including Turkey. Although most of the cases with Cx26 deafness have a recessive mode of inheritance, a small group of families demonstrated dominant or pseudodominant inheritance. In this report we present a Turkish family in which the proband had congenital profound deafness and was found to be homozygous for the 35delG mutation, whereas the father and a paternal uncle who had milder, late-onset sensorineural hearing loss had compound heterozygous 35delG and L90P mutations. This family and previous reports with the L90P mutation demonstrate that the hearing loss associated with the L90P/35delG genotype is consistently milder than that of 35delG homozygotes. GJB2 gene screening should be considered in families with seemingly dominant inheritance and late-onset moderate hearing loss.