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.     

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.     

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.     

Caution in interpretation of disease causality for heterozygous loss-of-function variants in the MYH8 gene associated with autosomal dominant disorder

To date, the NM_002472.2(MYH8):c.2021G>A (p.Arg674Gln) missense variant in the MYH8 gene is the only known genetic change in individuals with autosomal dominant trismus-pseudocamptodactyly syndrome with unknown molecular mechanism. Next-generation sequencing (NGS), including targeted gene panels and whole-exome sequencing, is routinely performed in many clinical diagnostic laboratories as standard-of-care testing aimed at identifying disease-causing genomic variants. Whole-exome sequencing has revealed loss-of-function variants in the MYH8 gene. To properly classify the MYH8 loss-of-function variants, we either retrieved them from public databases or retrospectively collected them from individuals genetically tested by custom NGS panels or by whole-exome sequencing and confirmed using Sanger sequencing. We further evaluated the respective clinical presentations of these individuals with the MYH8 loss-of-function variants. Heterozygous loss-of-function variants in the MYH8 gene were detected in 16 individuals without trismus-pseudocamptodactyly syndrome. Four of these 16 individuals had a pathogenic or likely pathogenic variant detected in another gene that could explain their clinical presentation. Moreover, there are ∼100 MYH8 heterozygous protein-truncating and splice site variants in the ExAC database in different populations. Our results, combined with the population data, indicate that loss-of-function variants in the MYH8 gene do not cause autosomal dominant trismus-pseudocamptodactyly syndrome, and the clinical significance of these variants remains unknown at present. This result highlights the importance of considering the molecular mechanism of disease, variants published in the medical literature, and population genomic data for the correct interpretation of loss-of-function variants in genes associated with autosomal dominant diseases.     

Revealing hidden genetic diagnoses in the ocular anterior segment disorders

PurposeOcular anterior segment disorders (ASDs) are clinically and genetically heterogeneous, and genetic diagnosis often remains elusive. In this study, we demonstrate the value of a combined analysis protocol using phenotypic, genomic, and pedigree structure data to achieve a genetic conclusion.MethodsWe utilized a combination of chromosome microarray, exome sequencing, and genome sequencing with structural variant and trio analysis to investigate a cohort of 41 predominantly sporadic cases.ResultsWe identified likely causative variants in 54% (22/41) of cases, including 51% (19/37) of sporadic cases and 75% (3/4) of cases initially referred as familial ASD. Two-thirds of sporadic cases were found to have heterozygous variants, which in most cases were de novo. Approximately one-third (7/22) of genetic diagnoses were found in rarely reported or recently identified ASD genes including PXDN, GJA8, COL4A1, ITPR1, CPAMD8, as well as the new phenotypic association of Axenfeld–Rieger anomaly with a homozygous ADAMTS17 variant. The remainder of the variants were in key ASD genes including FOXC1, PITX2, CYP1B1, FOXE3, and PAX6.ConclusionsWe demonstrate the benefit of detailed phenotypic, genomic, variant, and segregation analysis to uncover some of the previously “hidden” heritable answers in several rarely reported and newly identified ocular ASD-related disease genes.     

Computational biology insights into genotype-clinical phenotype-protein phenotype relationships between novel SLC26A2 variants identified in inherited skeletal dysplasias

BackgroundPathogenic variants in the transmembrane sulfate transporter protein SLC26A2 are associated with different phenotypes of inherited chondrodysplasias. As limited data is published from India, in this study we sought to elucidate the molecular basis of inherited chondrodysplasias in an Indian cohort.MethodsMolecular screening of 32 fetuses with antenatally diagnosed lethal skeletal dysplasia was performed by next generation sequencing and Sanger sequencing. The genotype-protein phenotype characterization was done using computational biology techniques like homology modelling, stability and pathogenicity predictions.ResultsWe identified five rare autosomal recessive SLC26A2 [NM_000112.4] variants, including three homozygous c.796dupA(p.Thr266Asnfs*12), c.1724delA(p.Lys575Serfs*10), and c.1375_1377dup(p.Val459dup) and two heterozygous variants (c.532C > T(p.Arg178*)) and (c.1382C > T(p.Ala461Val)) in compound heterozygous form in a total of four foetuses. Genotype-protein phenotype annotations highlighted that the clinically severe achondrogenesis 1B causative c.796dupA(p.Thr266Asnfs*12) and c.1724delA(p.Lys575Serfs*10)variants impact SLC26A2 protein structure by deletion of the protein core and transmembrane STAS domains, respectively. In clinically moderate atelosteogenesis type 2 phenotype, the c.1382C > T(p.Ala461Val) variant is predicted to distort alpha helix conformation and alter the bonding properties and free energy dynamics of transmembrane domains and the c.532C > T(p.Arg178*) variant results in loss of both core transmembrane and STAS domains of the SLC26A2 protein. The c.1375_1377dup(p.Val459dup) variant identified in clinically milder atelosteogenesis type II-diastrophic dysplasia spectrum lethal phenotype is predicted to decrease the Qualitative Model Energy Analysis (QMean), which affects major geometrical aspects of the SLC26A2 protein structure.ConclusionWe expand the spectrum of SLC26A2 related lethal chondrodysplasia and report three novel variants correlating clinical severity and protein phenotype within the lethal spectrum of this rare dysplasia. We demonstrate the relevance of structural characterization to aid novel variant reclassification to provide better prenatal management and reproductive options to families with lethal antenatal skeletal disorder.     

A comparative analysis of RAS variants in patients with disorders of somatic mosaicism

PurposeRAS genes (HRAS, KRAS, and NRAS) are commonly found to be mutated in cancers, and activating RAS variants are also found in disorders of somatic mosaicism (DoSM). A survey of the mutational spectrum of RAS variants in DoSM has not been performed.MethodsA total of 938 individuals with suspected DoSM underwent high-sensitivity clinical next-generation sequencing?based testing. We investigated the mutational spectrum and genotype?phenotype associations of mosaic RAS variants.ResultsIn this article, we present a series of individuals with DoSM with RAS variants. Classic hotspots, including Gly12, Gly13, and Gln61 constituted the majority of RAS variants observed in DoSM. Furthermore, we present 12 individuals with HRAS and KRAS in-frame duplication/insertion (dup/ins) variants in the switch II domain. Among the 18.3% individuals with RAS in-frame dup/ins variants, clinical findings were mainly associated with vascular malformations. Hotspots were associated with a broad phenotypic spectrum, including vascular tumors, vascular malformations, nevoid proliferations, segmental overgrowth, digital anomalies, and combinations of these. The median age at testing was higher and the variant allelic fraction was lower in individuals with in-frame dup/ins variants than those in individuals with mosaic RAS hotspots.ConclusionOur work provides insight into the allelic and clinical heterogeneity of mosaic RAS variants in nonmalignant conditions.     

Mutations of MYH14 are associated to anorectal malformations with recto‐perineal fistulas in a small subset of Chinese population

Background: Anorectal malformations (ARMs) are among the most commonly congenital abnormalities of distal hindgut development, ranging from anal stenosis to anal atresia with or without fistulas and persistent cloaca. The etiology remains elusive for most ARM cases and the majority of genetic studies on ARMs were based on a candidate gene approach. Materials and Methods: In all eight family members of a non‐consanguineous Chinese family, we performed whole‐exome sequencing. Subsequently, exome sequencing of MYH14 in 72 unrelated probands with ARMs was performed. The accurate distribution of non‐muscle myosin II heavy chain (NMHC II) was investigated by immunohistochemistry in serial sagittal sections of E11.5–13.5 mouse cloacal regions. Results: A homozygous mutation in MYH14 was identified in the two siblings of family 1. Compound heterozygous MYH14 changes were identified in an unrelated individual. Immunohistochemical analysis suggest stronger NMHC IIC localization in the epithelium of the murine embryonic cloaca, urorectal septum and hindgut compared with another two NMHC II isoforms. Conclusion: This is the first identification of mutations in MYH14 as a cause of ARMs. The stronger localization of NMHC IIC in E11.5–13.5 mouse cloacal regions further supports the role of MYH14 in anorectal development.     

Hand-foot-genital syndrome due to a duplication variant in the GC-rich region of HOXA13

BackgroundHand-Foot-Genital Syndrome (HFGS) is an autosomal dominant disorder characterized by a broad phenotypic spectrum. Variants in HOXA13 gene were associated with HFGS. To date, only twenty families with HFGS have been reported. However, the challenge in HFGS is the limited sample sizes and phenotypic heterogeneity. The advent of next-generation sequencing has permitted the identification of patients with HOXA13 variants who do not manifest with the full HFGS syndromic features.MethodsTrio (parents-proband) Whole-exome sequence(WES) and whole-genome sequencing(WGS) was carried out in this study to investigate the underlying pathogenic genetic factor of the neonate with a wide variety of clinical abnormalities.ResultsNo possible pathogenetic variation was detected by trio-WES, and a duplication variant in HOXA13 (c.360_377dup, p.Ala128_Ala133dup), inherited from her mother, was identified by the subsequent WGS in the proband with malnutrition, feeding difficulties, electrolyte disorders, metabolic acidosis, recurrent urinary tract infections, hydronephrosis, nephrolithiasis, abnormal ureter morphology, cholelithiasis, uterus didelphys. Sequence analysis of the variant region (exon1) indicated a high GC content of 73.92%. In addition, further enquiry of the family history revealed that 5 members of the family in 4 generations had hand and foot anomalies.ConclusionThe neonate was diagnosed with HFGS by genetic analysis. GC content had less influence on sequence coverage in WGS than WES analysis. This was the first report of trio-WGS study for HFGS genetic diagnosis, revealed that subsequent WGS was necessary for identification of potentially pathogenic variants in unexplained genetic disorders.     

Genome sequencing identifies multiple deleterious variants in autism patients with more severe phenotypes

PurposeTo maximize the discovery of potentially pathogenic variants to better understand the diagnostic utility of genome sequencing (GS) and to assess how the presence of multiple risk events might affect the phenotypic severity in autism spectrum disorders (ASD).MethodsGS was applied to 180 simplex and multiplex ASD families (578 individuals, 213 patients) with exome sequencing and array comparative genomic hybridization further applied to a subset for validation and cross-platform comparisons.ResultsWe found that 40.8% of patients carried variants with evidence of disease risk, including a de novo frameshift variant in NR4A2 and two de novo missense variants in SYNCRIP, while 21.1% carried clinically relevant pathogenic or likely pathogenic variants. Patients with more than one risk variant (9.9%) were more severely affected with respect to cognitive ability compared with patients with a single or no-risk variant. We observed no instance among the 27 multiplex families where a pathogenic or likely pathogenic variant was transmitted to all affected members in the family.ConclusionThe study demonstrates the diagnostic utility of GS, especially for multiple risk variants that contribute to the phenotypic severity, shows the genetic heterogeneity in multiplex families, and provides evidence for new genes for follow up.     

A homozygous loss-of-function variant in MYH11 in a case with megacystis-microcolon-intestinal hypoperistalsis syndrome

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is characterized by marked dilatation of the bladder and microcolon and decreased intestinal peristalsis. Recent studies indicate that heterozygous variants in ACTG2, which codes for a smooth muscle actin, cause MMIHS. However, such variants do not explain MMIHS cases that show an autosomal recessive mode of inheritance. We performed exome sequencing in a newborn with MMIHS and prune belly phenotype whose parents are consanguineous and identified a homozygous variant (c.3598A>T: p.Lys1200Ter) in MYH11, which codes for the smooth muscle myosin heavy chain. Previous studies showed that loss of Myh11 function in mice causes a bladder and intestinal phenotype that is highly reminiscent of MMIHS. All together, these observations strongly suggest that loss-of-function variants in MYH11 cause MMIHS. The documentation of variants in ACTG2 and MYH11 thus points to the involvement of the contractile apparatus of the smooth muscle in MMIHS. Interestingly, dominant-negative variants in MYH11 have previously been shown to cause thoracic aortic aneurism and dilatation. Different mechanisms of MYH11 disruption may thus lead to distinct patterns of smooth muscle dysfunction.     

Left ventricular non-compaction with Ebstein anomaly attributed to a TPM1 mutation

Left ventricular non-compaction (cardiomyopathy) (LVN(C)) is a rare hereditary cardiac condition, resulting from abnormal embryonic myocardial development. While it mostly occurs as an isolated condition, association with other cardiovascular manifestations such as Ebstein anomaly (EA) has been reported. This congenital heart defect is characterized by downward displacement of the tricuspid valve and leads to diminished ventricular size and function. In an autosomal dominant LVN(C) family consisting of five affected individuals, of which two also presented with EA and three with mitral valve insufficiency, we pursued the genetic disease cause using whole exome sequencing (WES). WES revealed a missense variant (p.Leu113Val) in TPM1 segregating with the LVN(C) phenotype. TPM1 encodes α-tropomyosin, which is involved in myocardial contraction, as well as in stabilization of non-muscle cytoskeletal actin filaments. So far, LVN(C)-EA has predominantly been linked to pathogenic variants in MYH7. However, one sporadic LVN(C)-EA case with a de novo TPM1 variant has recently been described. We here report the first LVN(C)-EA family segregating a pathogenic TPM1 variant, further establishing the association between EA predisposition and TPM1-related LVN(C). Consequently, we recommend genetic testing for both MYH7 and TPM1 in patients or families in which LVN(C)/non-compaction and EA coincide.     

Characterization of clinically relevant copy-number variants from exomes of patients with inherited heart disease and unexplained sudden cardiac death

PurposeCopy-number variant (CNV) analysis is increasingly performed in genetic diagnostics. We leveraged recent gene curation efforts and technical standards for interpretation and reporting of CNVs to characterize clinically relevant CNVs in patients with inherited heart disease and sudden cardiac death.MethodsExome sequencing data were analyzed for CNVs using eXome-Hidden Markov Model tool in 48 established disease genes. CNV breakpoint junctions were characterized. CNVs were classified using the American College of Medical Genetics and Genomics technical standards.ResultsWe identified eight CNVs in 690 unrelated probands (1.2%). Characterization of breakpoint junctions revealed nonhomologous end joining was responsible for four deletions, whereas one duplication was caused by nonallelic homologous recombination between duplicated sequences in MYH6 and MYH7. Identifying the precise breakpoint junctions determined the genomic involvement and proved useful for interpreting the clinical relevance of CNVs. Three large deletions involving TTN, MYBPC3, and KCNH2 were classified as pathogenic in three patients. Haplotype analysis of a deletion in ACTN2, found in two families, suggests the deletion was caused by an ancestral event.ConclusionCNVs infrequently cause inherited heart diseases and should be investigated when standard genetic testing does not reveal a genetic diagnosis.     

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder

PurposeDiphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).MethodsMolecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5^em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate–ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants.ResultsDPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate–ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.ConclusionWe provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.     

Further insights into the spectrum phenotype of TRAPPC9 and CDK5RAP2 genes,segregating independently in a large Tunisian family with intellectual disability and microcephaly

Intellectual disability (ID) often co-occurs with other neurologic phenotypes making molecular diagnosis more challenging particularly in consanguineous populations with the co-segregation of more than one ID-related gene in some cases.In this study, we investigated the phenotype of three patients from a large Tunisian family with significant ID phenotypic variability and microcephaly and performed a clinical exome sequencing in two cases. We identified, within the first branch, a homozygous variant in the TRAPPC9 gene (p.Arg472Ter) in two cases presenting severe ID, absent speech, congenital/secondary microcephaly in addition to autistic features, supporting the implication of TRAPPC9 in the “secondary” autism spectrum disorders and congenital microcephaly. In the second branch, we identified a homozygous variant (p.Lys189ArgfsTer15) in the CDK5RAP2 gene associated with an heterozygous TRAPPC9 variant (p.Arg472Ter) in one case harbouring primary hereditary microcephaly (MCPH) associated with an inter-hypothalamic adhesion, mixed hearing loss, selective thinning in the retinal nerve fiber layer and parafoveal ganglion cell complex, and short stature. Our findings expand the spectrum of the recently reported neurosensorial abnormalities and revealed the variable phenotype expressivity of CDK5RAP2 defect. Our study highlights the complexity of the genetic background of microcephaly/ID and the efficiency of the exome sequencing to provide an accurate diagnosis and to improve the management and follow-up of such patients.     

Integration of targeted sequencing and NIPT into clinical practice in a Chinese family with maple syrup urine disease

PurposeThis article demonstrates a prominent noninvasive prenatal approach to assist the clinical diagnosis of a single-gene disorder disease, maple syrup urine disease, using targeted sequencing knowledge from the affected family.MethodsThe method reported here combines novel mutant discovery in known genes by targeted massively parallel sequencing with noninvasive prenatal testing.ResultsBy applying this new strategy, we successfully revealed novel mutations in the gene BCKDHA (Ex2_4dup and c.392A>G) in this Chinese family and developed a prenatal haplotype-assisted approach to noninvasively detect the genotype of the fetus (transmitted from both parents).ConclusionThis is the first report of integration of targeted sequencing and noninvasive prenatal testing into clinical practice. Our study has demonstrated that this massively parallel sequencing–based strategy can potentially be used for single-gene disorder diagnosis in the future.     

Early-onset autosomal recessive cerebellar ataxia associated with retinal dystrophy: new human hotfoot phenotype caused by homozygous GRID2 deletion

PurposeThe aim of this study was to identify the genetic cause of early-onset autosomal recessive cerebellar ataxia associated with retinal dystrophy in a consanguineous family.MethodsAn affected 6-month-old child underwent neurological and ophthalmological examinations. Genetic analyses included homozygosity mapping, copy number analysis, conventional polymerase chain reaction, Sanger sequencing, quantitative polymerase chain reaction, and whole-exome sequencing. Expression analysis of GRID2 was performed by quantitative polymerase chain reaction and immunohistochemistry.ResultsA homozygous deletion of exon 2 of GRID2 (p.Gly30_Glu81del) was identified in the proband. GRID2 encodes an ionotropic glutamate receptor known to be selectively expressed in cerebellar Purkinje cells. Here, we demonstrated GRID2 expression in human adult retina and retinal pigment epithelium. In addition, Grid2 expression was demonstrated in different stages of murine retinal development. GRID2 immunostaining was shown in murine and human retina. Whole-exome sequencing in the proband did not provide arguments for other disease-causing mutations, supporting the idea that the phenotype observed represents a single clinical entity.ConclusionWe identified GRID2 as an underlying disease gene of early-onset autosomal recessive cerebellar ataxia with retinal dystrophy, expanding the clinical spectrum of GRID2 deletion mutants. We demonstrated for the first time GRID2 expression and localization in human and murine retina, providing evidence for a novel functional role of GRID2 in the retina.Genet Med17 4, 291–299.     

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.