May-Hegglin anomaly--from genome research to clinical laboratory

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly (MHA), Sebastian syndrome (SBS) and Fechtner syndrome (FTNS), are rare disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic D?hle body-like cytoplasmic inclusions in granulocytes. Epstein syndrome (EPS) is another autosomal dominant macrothrombocytopenia associated with Alport syndrome but without leukocyte inclusions. We previously mapped the locus for MHA on chromosome 22q12.3-q13.2 by genome-wide linkage analysis and found that MYH9, the gene for the nonmuscle myosin heavy chain-A (NMMHCA), is responsible for the disorder, by positional cloning. Mutations of MYH9 have also been found in SBS, FTNS, and EPS; the term "MYH9 disorders" has been proposed, but clear phenotype-genotype relationships were not found. We developed an immunofluorescence technique for conventional air-dried peripheral blood smears and studied the neutrophil NMMHCA localization in MYH9 disorders. Abnormal subcellular localization of NMMHCA was observed in every neutrophil from individuals with MYH9 mutations and the localization pattern was classified into three groups according to the number, size, and shape of the NMMHCA-positive granules. Patients without neutrophil inclusions on conventional May-Grünwald-Giemsa-stained blood smears but with abnormal NMMHCA localization on immunofluorescence analysis had MYH9 mutations. In contrast, patients with EPS and isolated macrothrombocytopenia with normal NMMHCA localization had no MYH9 mutations. Immunofluorescence analysis of neutrophil NMMHCA is useful as a novel screening test for the differential diagnosis of macrothrombocytopenia and clear hematopathological classification of MYH9 disorders.     

Pathogenetic mechanisms of hematological abnormalities of patients with MYH9 mutations

Mutations of MYH9, the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA), cause a complex clinical phenotype characterized by macrothrombocytopenia and granulocyte inclusion bodies, often associated with deafness, cataracts and/or glomerulonephritis. The pathogenetic mechanisms of these defects are either completely unknown or controversial. In particular, it is a matter of debate whether haploinsufficiency or a dominant-negative effect of mutant allele is responsible for hematological abnormalities. We investigated 11 patients from six pedigrees with different MYH9 mutations. We evaluated NMMHC-IIA levels in platelets and granulocytes isolated from peripheral blood and in megakaryocytes (Mks) cultured from circulating progenitors. NMMHC-IIA distribution in Mks and granulocytes was also assessed. We demonstrated that all the investigated patients had a 50% reduction of NMMHC-IIA expression in platelets and that a similar defect was present also in Mks. In subjects with R1933X and E1945X mutations, the whole NMMHC-IIA of platelets and Mks was wild-type. No NMMHC-IIA inclusions were observed at any time of Mk maturation. In granulocytes, the extent of NMMHC-IIA reduction in patients with respect to control cells was significantly greater than that measured in platelets and Mks, and we found that wild-type protein was sequestered within most of the NMMHC-IIA inclusions. Altogether these results indicate that haploinsufficiency of NMMHC-IIA in megakaryocytic lineage is the mechanism of macrothrombocytopenia consequent to MYH9 mutations, whereas in granulocytes a dominant-negative effect of mutant allele is involved in the formation of inclusion bodies. The finding that the same mutations act through different mechanisms in different cells is surprising and requires further investigation.     

Identification of six novel MYH9 mutations and genotype–phenotype relationships in autosomal dominant macrothrombocytopenia with leukocyte inclusions

The autosomal dominant macrothrombocytopenia with leukocyte inclusions, May-Hegglin anomaly (MHA), Sebastian syndrome (SBS), and Fechtner syndrome (FTNS), are rare platelet disorders characterized by a triad of giant platelets, thrombocytopenia, and characteristic Döhle body-like leukocyte inclusions. The locus for these disorders was previously mapped on chromosome 22q12.3–q13.2 and the disease gene was recently identified as MYH9, the gene encoding the nonmuscle myosin heavy chain-A. To elucidate the spectrum of MYH9 mutations responsible for the disorders and to investigate genotype–phenotype correlation, we examined MYH9 mutations in an additional 11 families and 3 sporadic patients with the disorders from Japan, Korea, and China. All 14 patients had heterozygous MYH9 mutations, including three known mutations and six novel mutations (three missense and three deletion mutations). Two cases had Alport manifestations including deafness, nephritis, and cataracts and had R1165C and E1841K mutations, respectively. However, taken together with three previous reports, including ours, the data do not show clear phenotype–genotype relationships. Thus, MHA, SBS, and FTNS appear to represent a class of allelic disorders with variable phenotypic diversity.     

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.     

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.     

MYH9-RD患者中性粒细胞包涵体的定位和本质鉴定

目的检测和分析非肌性肌球蛋白重链ⅡA(NMMHC-ⅡA)在MYH9-RD患者中性粒细胞胞浆中的定位及表达,以确定包涵体的构成本质。方法应用间接免疫荧光技术,观察MYH9-RD患者及正常对照者NMMHC-ⅡA在胞浆中的定位和分布情况;应用Western blot技术检测和分析中性粒细胞中NMMHC-ⅡA在患者和正常对照者之间蛋白表达水平的差异。结果免疫荧光技术清楚地显示了患者中性粒细胞胞浆中呈绿色荧光的"梭形"包涵体的形态和轮廓,与瑞特姬姆萨染色显示的包涵体形状、大小基本一致,但更为清晰;而正常对照者除有散点状的荧光斑点外,无包涵体形成;Western blot结果显示患者NMMHC-ⅡA表达量较正常对照者降低。结论 MYH9-RD患者中性粒细胞包涵体的主要成份是NMMHC-ⅡA,NMMHC-ⅡA的显阴性作用导致了包涵体的形成。     

A G to C transversion at the last nucleotide of exon 25 of the MYH9 gene results in a missense mutation rather than in a splicing defect

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils and might develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end-stage renal failure. In two families with macrothrombocytopenia we identified a novel c.3485G > C mutation in the last nucleotide of exon 25. Bioinformatic tools for splice site prediction and minigene functional test predicted splicing anomalies of exon 25. However, analysis of RNA purified from patient’s peripheral blood did not allowed us to detect any anomalies, suggesting that RNA processing is correct at least in this tissue. Therefore, we concluded that c.3485G > C leads to a novel missense mutation (p.Arg1162Thr) of myosin-9, which resulted to be slightly degraded in patient platelets. A precise definition of the effect of mutations is fundamental to improve our knowledge into the pathogenetic mechanisms responsible for the disease.     

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.     

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.     

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.     

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.     

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.     

A Trp33Arg mutation at exon 1 of the MYH9 gene in a Korean patient with May-Hegglin anomaly

In this report, we describe a Korean patient with May-Hegglin anomaly from a mutation of the MYH9 gene. The proband was a 21-year-old man with thrombocytopenia. He did not have a bleeding tendency. His neutrophil count was normal at 7490/mm3; however, the neutrophils contained abnormal basophilic inclusions in their cytoplasm. The platelet count was decreased at 15,000/mm3 with giant platelets. Coagulation test results were not remarkable. Direct sequencing of MYH9 revealed that he was heterozygous for a mutation in exon 1, which was a 97T>A substitution mutation affecting codon 33, substituting tryptophan with arginine (Trp33Arg). Family study showed that both of his parents had normal phenotype and genotypes, indicating a de novo occurrence of the mutation in the proband.     

A novel pathogenic MYH3 mutation in a child with Sheldon–Hall syndrome and vertebral fusions

Sheldon–Hall syndrome (SHS) is the most common of the distal arthrogryposes (DAs), a group of disorders characterized by congenital non‐progressive contractures. Patients with SHS present with contractures of the limbs and a distinctive triangular facies with prominent nasolabial folds. Calcaneovalgus deformity is frequent, as well as camptodactyly and ulnar deviation. Causative mutations in at least four different genes have been reported (MYH3, TNNI2, TPM2, and TNNT3). MYH3 plays a pivotal role in fetal muscle development and mutations in this gene are associated with Freeman–Sheldon syndrome, distal arthrogryposis 8 (DA8), and autosomal dominant spondylocarpotarsal synostosis. The last two disorders are characterized by skeletal abnormalities, in particular bony fusions. The observation that MYH3 may be mutated in these syndromes has suggested the involvement of this gene in bone development. We report the case of a boy with a novel pathogenic MYH3 mutation, presenting with the classical clinical features of SHS in association with unilateral carpal bone fusion and multiple vertebral fusions. This distinctive phenotype has never been reported in the literature so far and expands the phenotypic spectrum of SHS, endorsing the clinical variability of patients with MYH3‐related disorders. Our findings also support a role for MYH3 in both muscle and bone development, suggesting a phenotypic continuum in MYH3‐related disorders.

     

An MYH9 human disease model in flies: site-directed mutagenesis of the Drosophila non-muscle myosin II results in hypomorphic alleles with dominant character

We investigated whether or not human disease-causing, amino acid substitutions in MYH9 could cause dominant phenotypes when introduced into the sole non-muscle myosin II heavy chain in Drosophila melanogaster (zip/MyoII). We characterized in vivo the effects of four MYH9-like mutations in the myosin rod-R1171C, D1430N, D1847K and R1939X-which occur at highly conserved residues. These engineered mutant heavy chains resulted in D. melanogaster non-muscle myosin II with partial wild-type function. In a wild-type genetic background, mutant heavy chains were overtly recessive and hypomorphic: each was able to substitute partially for endogenous non-muscle myosin II heavy chain in animals lacking zygotically produced heavy chain (but the penetrance of rescue was below Mendelian expectation). Moreover, each of the four mutant heavy chains exhibits dominant characteristics when expressed in a sensitized genetic background (flies heterozygous for RhoA mutations). Thus, these zip/MyoII(MYH9) alleles function, like certain other hypomorphic alleles, as excellent bait in screens for genetic interactors. Our conjecture is that these mutations in D. melanogaster behave comparably to their parent mutations in humans. We further characterized these zip/MyoII(MYH9) alleles, and found that all were capable of correct spatial and temporal localization in animals lacking zygotic expression of wild-type zip/MyoII. In vitro, we demonstrate that mutant heavy chains can dimerize with endogenous, wild-type heavy chains, fold into coiled-coil structures and assemble into higher-order structures. Our work further supports D. melanogaster as a model system for investigating the basis of human disease.     

Attenuated familial adenomatous polyposis and Muir-Torre syndrome linked to compound biallelic constitutional MYH gene mutations

Attenuated familial adenomatous polyposis and Muir-Torre syndrome linked to compound biallelic constitutional MYH gene mutations.Peculiar dermatologic manifestations are present in several heritable gastrointestinal disorders. Muir-Torre syndrome (MTS) is a genodermatosis whose peculiar feature is the presence of sebaceous gland tumors associated with visceral malignancies. We describe one patient in whom multiple sebaceous gland tumors were associated with early onset colon and thyroid cancers and attenuated polyposis coli. Her family history was positive for colonic adenomas. She had a daughter presenting with yellow papules in the forehead region developed in the late infancy. Skin and visceral neoplasms were tested for microsatellite instability and immunohistochemical status of mismatch repair (MMR), APC and MYH proteins. The proband colon and skin tumors were microsatellite stable and showed normal expression of MMR proteins. Cytoplasmic expression of MYH protein was revealed in colonic cancer cells. Compound heterozygosity due to biallelic mutations in MYH, R168H and 379delC, was identified in the proband. The 11-year-old daughter was carrier of the monoallelic constitutional mutation 379delC in the MYH gene; in the sister, the R168H MYH gene mutation was detected. This report presents an interesting case of association between MYH-associated polyposis and sebaceous gland tumors. These findings suggest that patients with MTS phenotype that include colonic polyposis should be screened for MYH gene mutations.     

Mutations and sequence variation in the human myosin heavy chain IIa gene (MYH2)

We recently described a new autosomal dominant myopathy associated with a missense mutation in the myosin heavy chain (MyHC) IIa gene (MYH2). In this study, we performed mutation analysis of MYH2 in eight Swedish patients with familial myopathy of unknown cause. In two of the eight index cases, we identified novel heterozygous missense mutations in MYH2, one in each case: V970I and L1061V. The mutations were located in subfragment 2 of the MyHC and they changed highly conserved residues. Most family members carrying the mutations had signs and symptoms consisting mainly of mild muscle weakness and myalgia. In addition, we analyzed the extent and distribution of nucleotide variation in MYH2 in 50 blood donors, who served as controls, by the complete sequencing of all 38 exons comprising the coding region. We identified only six polymorphic sites, five of which were synonymous polymorphisms. One variant, which occurred at an allele frequency of 0.01, was identical to the L1061V that was also found in one of the families with myopathy. The results of the analysis of normal variation indicate that there is strong selective pressure against mutations in MYH2. On the basis of these results, we suggest that MyHC genes should be regarded as candidate genes in cases of hereditary myopathies of unknown etiology.     

Molecular analysis of the APC and MYH genes in Czech families affected by FAP or multiple adenomas: 13 novel mutations

Familial adenomatous polyposis (FAP) is an autosomal dominant predisposition to colorectal cancer and is caused by germline mutations in the adenomatous polyposis coli gene. The most prominent clinical manifestation is the presence of hundreds to thousands of colorectal polyps. A milder phenotype is found in patients affected with AFAP/ multiple adenomas. We screened the entire APC coding region using the combination of DGGE, PTT and direct sequencing and identified causative mutations in 52 of 77 patients. Thirteen of the mutations found were novel. In addition, we also tested 21 APC mutation/negative probands for the two most common mutations in the MYH gene. Four patients showed neither dominant transmission of the disease nor evidence of APC mutations. In one of them the most common biallelic germline mutation in the MYH gene was detected. Correlations between the localization of germline mutations and clinical manifestations of the diseases are discussed.     

Heterozygous variants in MYH10 associated with neurodevelopmental disorders and congenital anomalies with evidence for primary cilia-dependent defects in Hedgehog signaling

PurposeNonmuscle myosin II complexes are master regulators of actin dynamics that play essential roles during embryogenesis with vertebrates possessing 3 nonmuscle myosin II heavy chain genes, MYH9, MYH10, and MYH14. As opposed to MYH9 and MYH14, no recognizable disorder has been associated with MYH10. We sought to define the clinical characteristics and molecular mechanism of a novel autosomal dominant disorder related to MYH10.MethodsAn international collaboration identified the patient cohort. CAS9-mediated knockout cell models were used to explore the mechanism of disease pathogenesis.ResultsWe identified a cohort of 16 individuals with heterozygous MYH10 variants presenting with a broad spectrum of neurodevelopmental disorders and variable congenital anomalies that affect most organ systems and were recapitulated in animal models of altered MYH10 activity. Variants were typically de novo missense changes with clustering observed in the motor domain. MYH10 knockout cells showed defects in primary ciliogenesis and reduced ciliary length with impaired Hedgehog signaling. MYH10 variant overexpression produced a dominant-negative effect on ciliary length.ConclusionThese data presented a novel genetic cause of isolated and syndromic neurodevelopmental disorders related to heterozygous variants in the MYH10 gene with implications for disrupted primary cilia length control and altered Hedgehog signaling in disease pathogenesis.     

Germline MUTYH (MYH) mutations in Portuguese individuals with multiple colorectal adenomas

Germinal mutations in the base excision repair (BER) gene MUTYH (MYH) have recently been described in association with predisposition to multiple colorectal adenomas and cancer. In contrast to the classic dominant condition of familial adenomatous polyposis (FAP) due to germinal mutations in the APC gene, the MYH polyposis is an autosomal recessive disease. The identification of individuals affected by MYH polyposis brings new and important implications for the diagnostic, screening, genetic counseling, follow up and therapeutic options in these patients. In this study, screening for germinal mutations in the MYH gene was performed in 53 Portuguese individuals with multiple colorectal adenomas or classic adenomatous polyposis, in whom no mutation had been identified in the APC gene. The results revealed the presence of biallelic germline MYH mutations in 21 patients. In addition, we here report 3 mutations (c.340T>C [p.Y114H]; c.503G>A [p.R168H]; and c.1186_1187insGG [p.E396fsX437]) which, to our knowledge, have not been previously described.