Clinical and molecular genetic analysis of a family with macrothrombocytopenia and early onset sensorineural hearing loss

A kindred with inherited macrothrombocytopenia (MTCP) and sensorineural hearing loss (SNHL) from Ghent, Belgium was identified. Currently, joint expression of MTCP and hearing loss are linked to mutations within MYH9 only. Thus, we tested the hypothesis that a mutation within MYH9 is responsible for the autosomal dominant inheritance of MTCP and hearing loss in the Ghent family. A mutation screen of MYH9 coding region including its intron–exon junctions, as well as common hearing loss genes GJB2, GJB3, and GJB6, was performed. However, no pathogenic sequence alteration was identified. Patients' leukocytes were determined to be normal for NMMHC-A distribution via immunofluorescence analysis and free of Döhle body-like inclusions, identified as aggregates of mutant NMHC-IIA in MYH9 disorders. Also, western blot analysis with anti-NMHC-IIA antibody identified a single 220 kDa immunoreactive band with normal expression level of NMHC-IIA within the platelets and leukocytes of the affected family members. The immunoblot analysis eliminates the possibility of a large deletion within MYH9 that can escape detection by direct sequencing. Collectively, these results suggest that molecular genetic etiology of the Ghent family disorder may be due to as yet unidentified gene whose mutation(s) yields a phenocopy of the MYH9-related disease.     

两个有血缘关系的常染色体显性非综合性耳聋家系基因突变分析

目的 对两个有血缘关系的常染色体显性非综合性耳聋家系进行基因定位及突变分析,确定其致病基因.方法 通过家系调查和临床检查,鉴定了两个有血缘关系的常染色体显性非综合性耳聋大家系.并对已知位点及基因进行连锁分析,对致病基因在染色体上进行定位.PCR扩增候选基因MYH14基因的所有外显子和外显子-内含子交界区,直接测序法进行突变检测.结果 将这两个家系的致病基因定位于DFNA4位点,最大连锁值为4.94.具有统计学意义.突变检测发现MYH14基因的杂合突变c.359T>C(p.S120L),DNA直接测序确证两家系的所有患者均携带该突变,而家系中正常人则均不携带该突变.结论 第1次在中国非综合性耳聋家系中发现MYH14基因的突变,表明MYH14基因突变也是导致中国人非综合性耳聋的原因.     

MYH9相关综合征家系的临床表型和遗传学分析

目的 分析1组MYH9相关综合征家系的临床表现及遗传学特征.方法我们随访到1组4代51人的MYH9相关综合征家系,对目前存活的46人进行了临床表型和遗传学的初步分析.结果家系内有MYH9相关综合征患者17人,实验室检测都具有典型的"血小板减少、巨大血小板和粒细胞包涵体"三联症;临床表现具高度复杂性.并伴有严重的白血病、青光眼、转氨酶升高、血脂升高、哮喘、鼻炎及白内障等多种疾病,除此之外,本家系大部分感染者都有鼻炎和哮喘过敏史,而且当上述症状发作时,患者身上的出血点或紫癜会明显加重;在遗传方式上属于常染色体显性遗传.从细胞遗传学水平对家系中成员进行染色体检查,未发现核型异常.结论该MYH9相关综合征家系属常染色体显性遗传,染色体检查未发现核型异常;家系中的感染者不仅具有巨大血小板、血小板减少及中性粒细胞包涵体的特性,而且还具有严重的如:肝炎、白内障、白血痛、哮喘等临床表现.     

Identification of the first duplication in MYH9-related disease: A hot spot for unequal crossing-over within exon 24 of the MYH9 gene

MYH9-related disease (MYH9RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. All patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils. Some of them can also develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. The spectrum of mutations so far identified is peculiar, consisting of mostly missense mutations. Others are nonsense and frameshift mutations, all localized in the COOH terminus of the protein, or in-frame deletions. We report a family with three affected members carrying a novel mutation, the first duplication (p.E1066_A1072dup), of MYH9. The mutation was localized within exon 24, where the presence of a 16 nucleotide repeat was likely to be responsible for unequal crossing-over. Of note, a deletion of the same amino acids 1066_1072 was also identified in another MHY9RD family. Since two of the four patients with the duplication or the deletion in exon 24 were affected with bilateral neonatal cataracts, we speculate that these mutations might correlate with the ocular defect, which is reported only in 16% of MYH9RD patients.     

非综合征性耳聋一家系的基因定位

目的：定位1个一级表亲婚配非综合征性耳聋家系的致病基因，为分离该基因奠定基础。方法：先进行X染色体扫查，排除致病基因位于X染色体的可能；随后采用纯合子定位法，进行候选基因分析和常染色体基因组扫查；再对提示与致病基因紧密连锁的位点所在区域进一步分析，确定致病基因所在区域。结果：确认该家系的非综合征性耳聋为常染色体隐性遗传方式，候选基因分析排除25个已知基因是该家系致病基因的可能，而常染色体扫查提示致病基因位于D17S1293附近，进一步分析将其定位于D17S1850和D17S1818之间5．07cM区域。结论：该家系的致病基因定位于17q11.2-12的D17S1850和D17S1818之间5．07cM区域，是新的常染色体隐性遗传非综合征性耳聋致病基因位点。     

A complex phenotype of peripheral neuropathy, myopathy, hoarseness, and hearing loss is linked to an autosomal dominant mutation in MYH14

Both peripheral neuropathy and distal myopathy are well-established inherited neuromuscular disorders characterized by progressive weakness and atrophy of the distal limb muscles. A complex phenotype of peripheral neuropathy, myopathy, hoarseness, and hearing loss was diagnosed in a large autosomal dominant Korean family. A high density single nucleotide polymorphism (SNP)-based linkage study mapped the underlying gene to a region on chromosome 19q13.3. The maximum multipoint LOD score was 3.794. Sequencing of 34 positional candidate genes in the segregating haplotype revealed a novel c.2822G>T (p.Arg941Leu) mutation in the gene MYH14, which encodes the nonmuscle myosin heavy chain 14. Clinically we observed a sequential pattern of the onset of muscle weakness starting from the anterior to the posterior leg muscle compartments followed by involvement of intrinsic hand and proximal muscles. The hearing loss and hoarseness followed the onset of distal muscle weakness. Histopathologic and electrodiagnostic studies revealed both chronic neuropathic and myopathic features in the affected patients. Although mutations in MYH14 have been shown to cause nonsyndromic autosomal dominant hearing loss (DFNA4), the peripheral neuropathy, myopathy, and hoarseness have not been associated with MYH14. Therefore, we suggest that the identified mutation in MYH14 significantly expands the phenotypic spectrum of this gene.     

Genomic screening identifies novel linkages and provides further evidence for a role of MYH9 in nonsyndromic cleft lip and palate

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth anomaly that requires prolonged multidisciplinary rehabilitation. Although variation in several genes has been identified as contributing to NSCLP, most of the genetic susceptibility loci have yet to be defined. To identify additional contributory genes, a high-throughput genomic scan was performed using the Illumina Linkage IVb Panel platform. We genotyped 6008 SNPs in nine non-Hispanic white NSCLP multiplex families and a single large African-American NSCLP multiplex family. Fourteen chromosomal regions were identified with LOD>1.5, including six regions not previously reported. Analysis of the data from the African-American and non-Hispanic white families revealed two likely chromosomal regions: 8q21.3-24.12 and 22q12.2-12.3 with LOD scores of 2.98 and 2.66, respectively. On the basis of biological function, syndecan 2 (SDC2) and growth differentiation factor 6 (GDF6) in 8q21.3-24.12 and myosin heavy-chain 9, non-muscle (MYH9) in 22q12.2-12.3 were selected as candidate genes. Association analyses from these genes yielded marginally significant P-values for SNPs in SDC2 and GDF6 (0.01相似文献   

Coincidence of mutations in different connexin genes in Hungarian patients

Mutations in the GJB2 gene are the most common cause of hereditary prelingual sensorineural hearing impairment in Europe. Several studies indicate that different members of the connexin protein family interact to form gap junctions in the inner ear. Mutations in different connexin genes may accumulate and, consequently lead to hearing impairment. Therefore, we screened 47 Hungarian GJB2- heterozygous (one mutation in coding exon of the GJB2 gene) patients with hearing impairment for DNA changes in two further connexin genes (GJB6 and GJB3) and in the 5' non-coding region of GJB2 including the splice sites. Eleven out of 47 GJB2-heterozygous patients analyzed carried the splice site mutation -3170G>A in the 5'UTR region of GJB2. One out of these 11 patients showed homozygous -3170G>A genotype in combination with p.R127H. Next to the GJB2 mutations we noted 2 cases of deletion in GJB6 [Delta(GJB6-D13S1830)] and 3 (2 new and 1 described) base substitutions in GJB3 [c.357C>T, c.798C>T and c.94C>T (p.R32W)] which are unlikely disease-causing. Our results suggest the importance of routine screening for the rather frequent -3170G>A mutation (in addition to c.35delG) in patients with hearing impairment.     

Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus

About 60% of congenital hearing impairment cases in developed countries are due to genetic defects. Data on the molecular basis of hereditary hearing reflects vast genetic heterogeneity. There are >400 disorders in which hearing impairment is one of the characteristic traits of a syndrome. Linkage studies have identified more than 40 human chromosomal loci associated with non-syndromic hearing loss. So far, 16 of these 40 non-syndromic hearing impairment genes have been identified. We have studied the molecular basis of hearing impairment in four Druze families from the same village in Northern Galilee. The Druze are a small, isolated population in the Middle East practising endogamous marriage. Thus it was expected that a single mutation would account for hearing impairments in all these families. Our results show that at least four different genes are involved. Hearing impairment was caused in one family by a novel mutation in the recently identified OTOF (the DFNB9 gene), by a novel Pendred syndrome mutation (Thr193Ile) in another family, and by a GJB2 mutation (35delG also known as 30delG) in the third family. In the fourth family linkage was excluded from all known hearing impairments loci (recessive and dominant) as well as from markers covering chromosomes 11-22, pointing therefore to the existence of another non-syndromic recessive hearing loss (NSRD) locus on chromosomes 1-10.     

A novel nonsense mutation in MYO6 is associated with progressive nonsyndromic hearing loss in a Danish DFNA22 family

Autosomal dominant inheritance is described in about 20% of all nonsyndromic hearing loss with currently 54 distinct loci (DFNA1-54), and >20 different genes identified. Seven different unconventional myosin genes are involved in ten different types of syndromic and nonsyndromic hearing loss with different patterns of inheritance: MYO7A in DFNA11/DFNB2/USH1B, MYH9 in DFNA17, MYH14 in DFNA4, MYO6 in DFNA22/DFNB37, MYO3A in DFNB30, MYO1A in DFNA48, and MYO15A in DFNB3. Two missense mutations in MYO6 (p.C442Y and p.H246R) have been characterized in families of Italian and American Caucasian extraction with autosomal dominant hearing loss, respectively, and the latter was associated with cardiomyopathy in some patients. Three Pakistani families had homozygosity for three MYO6 mutations (c.36insT, p.R1166X, and p.E216V, respectively), and was in one instance associated with retinal degeneration. In the present study, we linked autosomal dominant hearing loss in a large Danish family to a 38.9 Mb interval overlapping with the DFNA22/DFNB37 locus on chromosome 6q13. A novel nonsense mutation in MYO6 exon 25 (c.2545C > T; p.R849X) was identified in the family. The mutation co-segregated with the disease and the mutant allele is predicted to encode a truncated protein lacking the coiled-coil and globular tail domains. These domains are hypothesized to be essential for targeting myosin VI to its cellular compartments. No other system was involved indicating nonsyndromic loss. In conclusion, a novel nonsense MYO6 mutation causes post-lingual, slowly progressive autosomal dominant nonsyndromic moderate to severe hearing loss in a Danish family.     

Non-muscle myosin heavy chain IIA and IIB interact and co-localize in living cells: relevance for MYH9-related disease

Myosins of class II constitute part of a superfamily of several classes of proteins expressed in almost all eukaryotic cell types. Differences in the heavy chains produce three isoforms of class II non-muscle myosins (A, B and C), which are widely distributed in most tissues and thought to be components of the cell motor systems, although specific functional roles are largely unknown. In particular, it is still a matter of debate whether they interact and have overlapping or distinct functions. This argument is relevant not only to cell physiology, but also to human pathology since mutations of the MYH9 gene encoding non-muscle myosin heavy chain II A (NMMHC-A) cause MYH9-related disease (MYH9-RD), an autosomal dominant disorder characterized by platelet macrocytosis, thrombocytopenia and leukocyte inclusions, variably associated with sensorineural hearing loss, cataracts and/or glomerulonephritis. In this study, we report the results of yeast two-hybrid screening showing that the C-terminals of NMMHC-A and -B interact. This interaction was confirmed by immunoprecipitation in transfected COS-7 cells and in skin fibroblasts naturally expressing both isoforms. Moreover, our immunomorphological study revealed that isoforms A and B co-localize in fibroblasts, erythroblasts and kidney cells. These results suggest that isoforms A and B are strictly related molecules and support the hypothesis that their interrelationship could be involved both in the variability of clinical phenotype and selectivity of tissue damage of MYH9-RD.     

Double heterozygosity for mutations in the beta-myosin heavy chain and in the cardiac myosin binding protein C genes in a family with hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy is a genetically heterogeneous autosomal dominant disease, caused by mutations in several sarcomeric protein genes. So far, seven genes have been shown to be associated with the disease with the beta-myosin heavy chain (MYH7) and the cardiac myosin binding protein C (MYBPC3) genes being the most frequently involved. We performed electrocardiography (ECG) and echocardiography in 15 subjects with hypertrophic cardiomyopathy from a French Caribbean family. Genetic analyses were performed on genomic DNA by haplotype analysis with microsatellite markers at each locus involved and mutation screening by single strand conformation polymorphism analysis. Based on ECG and echocardiography, eight subjects were affected and presented a classical phenotype of hypertrophic cardiomyopathy. Two new mutations cosegregating with the disease were found, one located in the MYH7 gene exon 15 (Glu483Lys) and the other in the MYBPC3 gene exon 30 (Glu1096 termination codon). Four affected subjects carried the MYH7 gene mutation, two the MYBPC3 gene mutation, and two were doubly heterozygous for the two mutations. The doubly heterozygous patients exhibited marked left ventricular hypertrophy, which was significantly greater than in the other affected subjects. We report for the first time the simultaneous presence of two pathological mutations in two different genes in the context of familial hypertrophic cardiomyopathy. This double heterozygosity is not lethal but is associated with a more severe phenotype.     

A novel mutation in the mitochondrial tRNA(Ser(UCN)) gene in a family with non-syndromic sensorineural hearing impairment

We describe a family with non-syndromic sensorineural hearing impairment inherited in a manner consistent with maternal transmission. Affected members were found to have a novel heteroplasmic mtDNA mutation, T7510C, in the tRNA^Ser(UCN) gene. This mutation was not found in 661 controls, is well conserved between species, and disrupts base pairing in the acceptor stem of the tRNA, making it the probable cause of hearing impairment in this family. Sequencing of the other mitochondrial tRNA genes did not show any other pathogenic mutations. Four other mutations causing hearing impairment have been reported in the tRNA^Ser(UCN) gene, two having been shown to affect tRNA^Ser(UCN) levels. With increasing numbers of reports of mtDNA mutations causing hearing impairment, screening for such mutations should be considered in all cases unless mitochondrial inheritance can be excluded for certain.


Keywords: hearing impairment; mtDNA mutation; tRNA^Ser(UCN)     

MYH9-related disease: Five novel mutations expanding the spectrum of causative mutations and confirming genotype/phenotype correlations

MYH9-related disease (MYH9-RD) is a rare autosomal dominant syndromic disorder caused by mutations in MYH9, the gene encoding for the heavy chain of non-muscle myosin IIA (myosin-9). MYH9-RD is characterized by congenital macrothrombocytopenia and typical inclusion bodies in neutrophils associated with a variable risk of developing sensorineural deafness, presenile cataract, and/or progressive nephropathy. The spectrum of mutations responsible for MYH9-RD is limited. We report five families, each with a novel MYH9 mutation. Two mutations, p.Val34Gly and p.Arg702Ser, affect the motor domain of myosin-9, whereas the other three, p.Met847_Glu853dup, p.Lys1048_Glu1054del, and p.Asp1447Tyr, hit the coiled-coil tail domain of the protein. The motor domain mutations were associated with more severe clinical phenotypes than those in the tail domain.     

Grhl2 deficiency impairs otic development and hearing ability in a zebrafish model of the progressive dominant hearing loss DFNA28

Congenital and progressive hearing impairment is a common distressing disease. The progressive dominant hearing loss DFNA28 in human is associated with a frameshift mutation of Grainyhead-like 2 (GRHL2) but its etiology and mechanism remain unknown. Here we report a zebrafish grhl2b(T086) mutant line in which grhl2b expression is interrupted by an insertion of a Tol2 transposon element. The mutants exhibit enlarged otocysts, smaller or eliminated otoliths, malformed semicircular canals, insensitiveness to sound stimulation and imbalanced swimming motion. Since grainyhead-like family members can regulate epithelial adhesion, we examined the expression of some genes encoding junction proteins in mutants. We show that the expression of claudin b (cldnb) and epcam is abolished or dramatically reduced and apical junctional complexes are abnormal in otic epithelial cells of mutant embryos. Co-injection of cldnb and epcam mRNA could largely rescue the mutant phenotype. Injection of human wild-type GRHL2 mRNA but not the mutant GRHL2 mRNA derived from DFNA28 patients into grhl2b(T086) mutant embryos could rescue the inner-ear defects. Furthermore, we demonstrate that Grhl2b directly binds to the enhancers and promotes the expression of cldnb and epcam. Thus, this work reveals an evolutionarily conserved function of Grhl2 in otic development and provides a fish model for further studying mechanisms of Grhl2-related hearing loss.     

Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease. We have evaluated 108 consecutive MYH9-RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases). We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients' clinical management but also to the elucidation of the pathogenesis of the disease.     

Next‐generation sequencing for the diagnosis of MYH9‐RD: Predicting pathogenic variants

The heterogeneous manifestations of MYH9‐related disorder (MYH9‐RD), characterized by macrothrombocytopenia, Döhle‐like inclusion bodies in leukocytes, bleeding of variable severity with, in some cases, ear, eye, kidney, and liver involvement, make the diagnosis for these patients still challenging in clinical practice. We collected phenotypic data and analyzed the genetic variants in more than 3,000 patients with a bleeding or platelet disorder. Patients were enrolled in the BRIDGE‐BPD and ThromboGenomics Projects and their samples processed by high throughput sequencing (HTS). We identified 50 patients with a rare variant in MYH9. All patients had macrothrombocytes and all except two had thrombocytopenia. Some degree of bleeding diathesis was reported in 41 of the 50 patients. Eleven patients presented hearing impairment, three renal failure and two elevated liver enzymes. Among the 28 rare variants identified in MYH9, 12 were novel. HTS was instrumental in diagnosing 23 patients (46%). Our results confirm the clinical heterogeneity of MYH9‐RD and show that, in the presence of an unclassified platelet disorder with macrothrombocytes, MYH9‐RD should always be considered. A HTS‐based strategy is a reliable method to reach a conclusive diagnosis of MYH9‐RD in clinical practice.     

遗传性耳聋检测及风险分析

遗传性耳聋由遗传缺陷引起,与基因异常及相关的综合症关系密切,属临床常见疾病。鉴于目前绝大多数的遗传性耳聋无有效治疗手段,遗传咨询的意义显得尤为重要,其核心问题为遗传风险的估算。简要介绍了遗传性耳聋的分类以及利用致病基因特点和家系信息进行概率分析的方法。     

Involvement of DFNB59 mutations in autosomal recessive nonsyndromic hearing impairment

In a consanguineous Turkish family, a locus for autosomal recessive nonsyndromic hearing impairment (ARNSHI) was mapped to chromosome 2q31.1-2q33.1. Microsatellite marker analysis in the complete family determined the critical linkage interval that overlapped with DFNB27, for which the causative gene has not yet been identified, and DFNB59, a recently described auditory neuropathy caused by missense mutations in the DFNB59 gene. The 352-amino acid (aa) DFNB59 gene product pejvakin is present in hair cells, supporting cells, spiral ganglion cells, and the first three relays of the afferent auditory pathway. A novel homozygous nonsense mutation (c.499C>T; p.R167X) was detected in the DFNB59 gene, segregating with the deafness in the family. The mRNA derived from the mutant allele was found not to be degraded in lymphocytes, indicating that a truncated pejvakin protein of 166 aa may be present in the affected individuals. Screening of 67 index patients from additional consanguineous Turkish families with autosomal recessive hearing impairment revealed a homozygous missense mutation (c.547C>T; p.R183W) that segregates with the hearing impairment in one family. Furthermore, in a panel of 83 Dutch patients, two additional novel mutations (c.509_512delCACT; p.S170CfsX35 and c.731T>G; p.L244R), which were not present in ethnically matched controls, were found heterozygously. Together, our data indicate that also nonsense mutations in DFNB59 cause nonsyndromic hearing loss, but that mutations in DFNB59 are not a major cause of nonsyndromic hearing impairment in the Turkish and Dutch population.