Differential diagnosis of vacuolar myopathies in the NGS era

Altered autophagy accompanied by abnormal autophagic (rimmed) vacuoles detectable by light and electron microscopy is a common denominator of many familial and sporadic non‐inflammatory muscle diseases. Even in the era of next generation sequencing (NGS), late‐onset vacuolar myopathies remain a diagnostic challenge. We identified 32 adult vacuolar myopathy patients from 30 unrelated families, studied their clinical, histopathological and ultrastructural characteristics and performed genetic testing in index patients and relatives using Sanger sequencing and NGS including whole exome sequencing (WES). We established a molecular genetic diagnosis in 17 patients. Pathogenic mutations were found in genes typically linked to vacuolar myopathy (GNE, LDB3/ZASP, MYOT, DES and GAA), but also in genes not regularly associated with severely altered autophagy (FKRP, DYSF, CAV3, COL6A2, GYG1 and TRIM32) and in the digenic facioscapulohumeral muscular dystrophy 2. Characteristic histopathological features including distinct patterns of myofibrillar disarray and evidence of exocytosis proved to be helpful to distinguish causes of vacuolar myopathies. Biopsy validated the pathogenicity of the novel mutations p.(Phe55*) and p.(Arg216*) in GYG1 and of the p.(Leu156Pro) TRIM32 mutation combined with compound heterozygous deletion of exon 2 of TRIM32 and expanded the phenotype of Ala93Thr‐caveolinopathy and of limb‐girdle muscular dystrophy 2i caused by FKRP mutation. In 15 patients no causal variants were detected by Sanger sequencing and NGS panel analysis. In 12 of these cases, WES was performed, but did not yield any definite mutation or likely candidate gene. In one of these patients with a family history of muscle weakness, the vacuolar myopathy was eventually linked to chloroquine therapy. Our study illustrates the wide phenotypic and genotypic heterogeneity of vacuolar myopathies and validates the role of histopathology in assessing the pathogenicity of novel mutations detected by NGS. In a sizable portion of vacuolar myopathy cases, it remains to be shown whether the cause is hereditary or degenerative.     

Autoimmunity in a cohort of 471 patients with primary antibody deficiencies

Objectives: The aim of this study was to evaluate the frequency of autoimmunity in primary antibody deficiency (PAD).

Methods: A total of 471 patients with PADs enrolled in this retrospective cohort study. For all patients’ demographic information, clinical records and laboratory data were collected to investigate autoimmune complications.

Results: Autoimmune disorders as the first presentation of immunodeficiency were recorded in 11 patients (2.5%). History of autoimmunity was recorded in 125 patients during the course of the disease (26.5%). The frequency of autoimmunity in common variable immune deficiency (32.0%) was higher than other forms of PADs. The most common autoimmune manifestations were reported to be autoimmune gastrointestinal disease and autoimmune cytopenias. Among patients with autoimmunity, 87 patients (69.6%) had a history of one autoimmune disorder, while 38 patients (30.4%) had a history of multiple autoimmunities. The immune thrombocytopenic purpura and autoimmune hemolytic anemia were the most two concomitant autoimmune disorders in 16 (42.1%) of 38 patients with multiple autoimmunities. Comparing the frequency of Tregs in PAD patients with autoimmunity showed that, patients with multiple autoimmunities had lower Tregs than those with single autoimmunity (p = 0.017).

Conclusion: It is important that non-immunologist physicians be alert of the associated autoimmunity with PADs in order to reduce the diagnostic delay and establish timely immunoglobulin replacement therapy in these patients.     

Targeted and Genomewide NGS Data Disqualify Mutations in MYO1A,the “DFNA48 Gene”, as a Cause of Deafness

MYO1A is considered the gene underlying autosomal dominant nonsyndromic hearing loss DFNA48, based on six missense variants, one small in‐frame insertion, and one nonsense mutation. Results from NGS targeting 66 deafness genes in 109 patients identified three families challenging this assumption: two novel nonsense (p.Tyr740* and p.Arg262*) and a known missense variant were identified heterozygously not only in index patients, but also in unaffected relatives. Deafness in these families clearly resulted from mutations in other genes (MYO7A, EYA1, and CIB2). Most of the altogether 10 MYO1A mutations are annotated in dbSNP, and population frequencies (dbSNP, 1000 Genomes, Exome Sequencing Project) above 0.1% contradict pathogenicity under a dominant model. One healthy individual was even homozygous for p.Arg262*, compatible with homozygous Myo1a knockout mice lacking any overt pathology. MYO1A seems dispensable for hearing and overall nonessential. MYO1A adds to the list of “erroneous disease genes”, which will expand with increasing availability of large‐scale sequencing data.     

Utility of next‐generation sequencing technologies for the efficient genetic resolution of haematological disorders

Next‐generation sequencing (NGS) has now evolved to be a relatively affordable and efficient means of detecting genetic mutations. Whole genome sequencing (WGS) or whole exome sequencing (WES) offers the opportunity for rapid diagnosis in many paediatric haematological conditions, where phenotypes are variable and either a large number of genes are involved, or the genes are large making sanger sequencing expensive and labour‐intensive. NGS offers the potential for gene discovery in patients who do not have mutations in currently known genes. This report shows how WES was used in the diagnosis of six paediatric haematology cases. In four cases (Diamond–Blackfan anaemia, congenital neutropenia (n = 2), and Fanconi anaemia), the diagnosis was suspected based on classical phenotype, and NGS confirmed those suspicions. Mutations in RPS19, ELANE and FANCD2 were found. The final two cases (MYH9 associated macrothrombocytopenia associated with multiple congenital anomalies; atypical juvenile myelomonocytic leukaemia associated with a KRAS mutation) highlight the utility of NGS where the diagnosis is less certain, or where there is an unusual phenotype. We discuss the advantages and limitations of NGS in the setting of these cases, and in haematological conditions more broadly, and discuss where NGS is most efficiently used.     

Association of IGHM polymorphisms with susceptibility to type 1 diabetes

Differentiation of B lymphocytes is accompanied by a regulated switch in the expression pattern and stability of surface and secretory immunoglobulins (Igs). Several lines of evidence show that autoimmune responses evolving in much autoimmune pathologies were associated with a high level of humoral Ig, but their pathogenic role remains elusive. The aim of this study was to test the hypothesis that variants at the immunoglobulin heavy-chain IGH locus are genetic determinants to T1D susceptibility. Here, we tested the genetic association of the variants of the immunoglobulin heavy-chain IGH locus as a genetic determinant to T1D susceptibility. A total of 255 subjects from 59 Tunisian families were genotyped for 15 SNPs mapping in 4 regions in IGH locus. We found that rs1950942, rs2180790, rs1808152, and rs1956596 of IGHM and rs2516751 variant located in the IGHA1/IGHG2 region were significantly associated with a risk for T1D p?=?7E-3; p?=?0.03; p?=?0.02; p?=?0.043; and p?=?3.65E-5, respectively. The TATGG haplotype derived from LD across three SNPs from IGHM gene and two SNPs from IGHD gene was significantly over-transmitted from parents to affect offspring. Our results suggest that genetic variants at the IGH locus are associated with T1D susceptibility. These variations may predispose to IgG AutoAbs production against pancreatic antigens and AutoAbs multi-reactivity, leading to T1D development.

     

Targeted next‐generation sequencing and parental genotyping in sporadic Chinese Han deaf patients

The interpretation of the targeted next‐generation sequencing (NGS) results can be challenging for variants identified in the sporadic deaf patients. In this study, we performed targeted NGS of 143 deafness‐associated genes in 44 sporadic deaf patients and use parental genotyping to test whether the candidate pathogenic variants complied with recessive or de novo pattern. Of 29 recessive candidate variants with minor allele frequencies (MAFs) less than 0.005, 3 pairs of apparent compound heterozygous variants were inherited from the same parental allele, ruling out their pathogenic roles. In addition, non‐segregation of an OTOA p.Gln293Arg variant led to the discovery of a genomic microdeletion of OTOA on the opposite allele by copy number variation analysis. Overall, 13 pairs of recessive candidate variants were deemed causative in 13 patients. Of the 28 dominant candidate variants with MAFs less than 0.0005, none occurred de novo, suggesting that they were not disease causing. Our results revealed that targeted NGS in sporadic deaf patients may generate a significant false‐positive rate. Parental genotyping is a simple but effective step toward minimizing the false‐positive results. Our study also showed that de novo variants in dominant deafness genes may not be a common cause for sporadic deafness.     

NGS testing for cardiomyopathy: Utility of adding RASopathy‐associated genes

RASopathies include a group of syndromes caused by pathogenic germline variants in RAS‐MAPK pathway genes and typically present with facial dysmorphology, cardiovascular disease, and musculoskeletal anomalies. Recently, variants in RASopathy‐associated genes have been reported in individuals with apparently nonsyndromic cardiomyopathy, suggesting that subtle features may be overlooked. To determine the utility and burden of adding RASopathy‐associated genes to cardiomyopathy panels, we tested 11 RASopathy‐associated genes by next‐generation sequencing (NGS), including NGS‐based copy number variant assessment, in 1,111 individuals referred for genetic testing for hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). Disease‐causing variants were identified in 0.6% (four of 692) of individuals with HCM, including three missense variants in the PTPN11, SOS1, and BRAF genes. Overall, 36 variants of uncertain significance (VUSs) were identified, averaging ～3VUSs/100 cases. This study demonstrates that adding a subset of the RASopathy‐associated genes to cardiomyopathy panels will increase clinical diagnoses without significantly increasing the number of VUSs/case.     

Next‐generation DNA sequencing of a Swedish malignant hyperthermia cohort

Malignant hyperthermia (MH)‐related mutations have been identified in the ryanodine receptor type 1 gene (RYR1) and in the dihydropyridine gene (CACNA1S), but about half of the patients do not have causative mutations in these genes. We wanted to study the contribution of other muscle genes to the RYR1 phenotypes. We designed a gene panel for sequence enrichment targeting 64 genes of proteins involved in the homeostasis of the striated muscle cell. Next‐generation sequencing (NGS) resulted in >50,000 sequence variants which were further analyzed by software filtering criteria to identify causative variants. In four of five patients we identified previously reported RYR1 mutations while the fifth patient did not show any candidate variant in any of the genes investigated. In two patients pathogenic variants were found in other genes known to cause a muscle disorders. All but one patient carried likely benign rare polymorphisms. The NGS technique proved convenient in identifying variants in the RYR1. However, with a clinically variable phenotype‐like MH, the pre‐selection of genes poses problems in variant interpretation.     

Rett‐like phenotypes: expanding the genetic heterogeneity to the KCNA2 gene and first familial case of CDKL5‐related disease

Several genes have been implicated in Rett syndrome (RTT) in its typical and variant forms. We applied next‐generation sequencing (NGS) to evaluate for mutations in known or new candidate genes in patients with variant forms of Rett or Rett‐like phenotypes of unknown molecular aetiology. In the first step, we used NGS with a custom panel including MECP2, CDKL5, FOXG1, MEF2C and IQSEC2. In addition to a FOXG1 mutation in a patient with all core features of the congenital variant of RTT, we identified a missense (p.Ser240Thr) in CDKL5 in a patient who appeared to be seizure free. This missense was maternally inherited with opposite allele expression ratios in the proband and her mother. In the asymptomatic mother, the mutated copy of the CDKL5 gene was inactivated in 90% of blood cells. We also identified a premature stop codon (p.Arg926*) in IQSEC2 in a patient with a Rett‐like phenotype. Finally, exome sequencing enabled us to characterize a heterozygous de novo missense (p.Val408Ala) in KCNA2 encoding the potassium channel Kv 1.2 in a girl with infantile‐onset seizures variant of RTT. Our study expands the genetic heterogeneity of RTT and RTT‐like phenotypes. Moreover, we report the first familial case of CDKL5‐related disease.     

Is gene panel sequencing more efficient than clinical‐based gene sequencing to diagnose autoinflammatory diseases? A randomized study

The aim of this study was to compare the effectiveness of the gene‐panel next‐generation sequencing (NGS) strategy versus the clinical‐based gene Sanger sequencing for the genetic diagnosis of autoinflammatory diseases (AIDs). Secondary goals were to describe the gene and mutation distribution in AID patients and to evaluate the impact of the genetic report on the patient’s medical care and treatment. Patients with AID symptoms were enrolled prospectively and randomized to two arms, NGS (n = 99) (32–55 genes) and Sanger sequencing (n = 197) (one to four genes). Genotypes were classified as ‘consistent/confirmatory’, ‘uncertain significance’ or ‘non‐contributory’. The proportion of patients with pathogenic genotypes concordant with the AID phenotype (consistent/confirmatory) was significantly higher with NGS than Sanger sequencing [10 of 99 (10·1%) versus eight of 197 (4·1%)]. MEFV, ADA2 and MVK were the most represented genes with a consistent/confirmed genotype, whereas MEFV, NLRP3, NOD2 and TNFRSF1A were found in the ‘uncertain significance’ genotypes. Six months after the genetic report was sent, 54 of 128 (42·2%) patients had received effective treatment for their symptoms; 13 of 128 (10·2%) had started treatment after the genetic study. For 59 of 128 (46%) patients, the results had an impact on their overall care, independent of sequencing group and diagnostic conclusion. Targeted NGS improved the diagnosis and global care of patients with AIDs.     

The next generation of population-based spinal muscular atrophy carrier screening: comprehensive pan-ethnic SMN1 copy-number and sequence variant analysis by massively parallel sequencing

PurposeTo investigate pan-ethnic SMN1 copy-number and sequence variation by hybridization-based target enrichment coupled with massively parallel sequencing or next-generation sequencing (NGS).MethodsNGS reads aligned to SMN1 and SMN2 exon 7 were quantified to determine the total combined copy number of SMN1 and SMN2. The ratio of SMN1 to SMN2 was calculated based on a single-nucleotide difference that distinguishes the two genes. SMN1 copy-number results were compared between the NGS and quantitative polymerase chain reaction and/or multiplex ligation-dependent probe amplification. The NGS data set was also queried for the g.27134T>G single-nucleotide polymorphism (SNP) and other SMN1 sequence pathogenic variants.ResultsThe sensitivity of the test to detect spinal muscular atrophy (SMA) carriers with one copy of SMN1 was 100% (95% confidence interval (CI): 95.9–100%; n = 90) and specificity was 99.6% (95% CI: 99.4–99.7%; n = 6,648). Detection of the g.27134T>G SNP by NGS was 100% concordant with an restriction fragment-length polymorphism method (n = 493). Ten single-nucleotide variants in SMN1 were detectable by NGS and confirmed by gene-specific amplicon-based sequencing. This comprehensive approach yielded SMA carrier detection rates of 90.3–95.0% in five ethnic groups studied.ConclusionWe have developed a novel, comprehensive SMN1 copy-number and sequence variant analysis method by NGS that demonstrated improved SMA carrier detection rates across the entire population examined.Genet Med advance online publication 19 January 2017     

WDR45B‐related intellectual disability,spastic quadriplegia,epilepsy, and cerebral hypoplasia: A consistent neurodevelopmental syndrome

The advancement in genomic sequencing has greatly improved the diagnostic yield for neurodevelopmental disorders and led to the discovery of large number of novel genes associated with these disorders. WDR45B has been identified as a potential intellectual disability gene through genomic sequencing of 2 large cohorts of affected individuals. In this report we present 6 individuals from 3 unrelated families with homozygous pathogenic variants in WDR45B: c.799C>T (p.Q267*) in 1 family and c.673C>T (p.R225*) in 2 families. These individuals shared a similar phenotype including profound development delay, early‐onset refractory epilepsy, progressive spastic quadriplegia and contractures, and brain malformations. Neuroimaging showed ventriculomegaly, reduced cerebral white matter volume, and thinning of cerebral gray matter. The consistency in the phenotype strongly supports that WDR45B is associated with this disease.     

Genetic and clinical findings in a Chinese cohort of patients with collagen VI‐related myopathies

Collagen VI‐related myopathy, caused by pathogenic variants in the genes encoding collagen VI, represents a clinical continuum from Ullrich congenital muscular dystrophy (UCMD) to Bethlem myopathy (BM). Clinical data of 60 probands and their family members were collected and muscle biopsies of 26 patients were analyzed. COL6A1, COL6A2 and COL6A3 exons were analyzed by direct sequencing or next generation sequencing (NGS). Sixty patients were characterized by delayed motor milestones, muscle weakness, skin and joint changes with 40 UCMD and 20 BM. Muscle with biopsies revealed dystrophic changes and showed completely deficiency of collagen VI or sarcolemma specific collagen VI deficiency. We identified 62 different pathogenic variants in these 60 patients, with 34 were first reported while 28 were previously known; 72 allelic pathogenic variants in COL6A1 (25/72, 34.7%), COL6A2 (33/72, 45.8%) and COL6A3 (14/72, 19.4%). We also found somatic mosaic variant in the parent of 1 proband by personal genome machine amplicon deep sequencing for mosaicism. Here we provide clinical, histological and genetic evidence of collagen VI‐related myopathy in 60 Chinese patients. NGS is a valuable approach for diagnosis and accurate diagnosis provides useful information for genetic counseling of related families.     

Molecular and phenotypic spectrum of Noonan syndrome in Chinese patients

Noonan syndrome (NS) is a common autosomal dominant/recessive disorder. No large-scale study has been conducted on NS in China, which is the most populous country in the world. Next-generation sequencing (NGS) was used to identify pathogenic variants in patients that exhibited NS-related phenotypes. We assessed the facial features and clinical manifestations of patients with pathogenic or likely pathogenic variants in the RAS-MAPK signaling pathway. Gene-related Chinese NS facial features were described using artificial intelligence (AI).NGS identified pathogenic variants in 103 Chinese patients in eight NS-related genes: PTPN11 (48.5%), SOS1 (12.6%), SHOC2 (11.7%), KRAS (9.71%), RAF1 (7.77%), RIT1 (6.8%), CBL (0.97%), NRAS (0.97%), and LZTR1 (0.97%). Gene-related facial representations showed that each gene was associated with different facial details. Eight novel pathogenic variants were detected and clinical features because of specific genetic variants were reported, including hearing loss, cancer risk due to a PTPN11 pathogenic variant, and ubiquitous abnormal intracranial structure due to SHOC2 pathogenic variants. NGS facilitates the diagnosis of NS, especially for patients with mild/moderate and atypical symptoms. Our study describes the genotypic and phenotypic spectra of NS in China, providing new insights into distinctive clinical features due to specific pathogenic variants.     

Development of an evidence-based algorithm that optimizes sensitivity and specificity in ES-based diagnostics of a clinically heterogeneous patient population

PurposeNext-generation sequencing (NGS) is rapidly replacing Sanger sequencing in genetic diagnostics. Sensitivity and specificity of NGS approaches are not well-defined, but can be estimated from applying NGS and Sanger sequencing in parallel. Utilizing this strategy, we aimed at optimizing exome sequencing (ES)–based diagnostics of a clinically diverse patient population.MethodsConsecutive DNA samples from unrelated patients with suspected genetic disease were exome-sequenced; comparatively nonstringent criteria were applied in variant calling. One thousand forty-eight variants in genes compatible with the clinical diagnosis were followed up by Sanger sequencing. Based on a set of variant-specific features, predictors for true positives and true negatives were developed.ResultsSanger sequencing confirmed 81.9% of ES-derived variants. Calls from the lower end of stringency accounted for the majority of the false positives, but also contained ~5% of the true positives. A predictor incorporating three variant-specific features classified 91.7% of variants with 100% specificity and 99.75% sensitivity. Confirmation status of the remaining variants (8.3%) was not predictable.ConclusionsCriteria for variant calling in ES-based diagnostics impact on specificity and sensitivity. Confirmatory sequencing for a proportion of variants, therefore, remains a necessity. Our study exemplifies how these variants can be defined on an empirical basis.     

A concurrent dual analysis of genomic data augments diagnoses: Experiences of 2 clinical sites in the Undiagnosed Diseases Network

PurposeNext-generation sequencing (NGS) has revolutionized the diagnostic process for rare/ultrarare conditions. However, diagnosis rates differ between analytical pipelines. In the National Institutes of Health-Undiagnosed Diseases Network (UDN) study, each individual’s NGS data are concurrently analyzed by the UDN sequencing core laboratory and the clinical sites. We examined the outcomes of this practice.MethodsA retrospective review was performed at 2 UDN clinical sites to compare the variants and diagnoses/candidate genes identified with the dual analyses of the NGS data.ResultsIn total, 95 individuals had 100 diagnoses/candidate genes. There was 59% concordance between the UDN sequencing core laboratories and the clinical sites in identifying diagnoses/candidate genes. The core laboratory provided more diagnoses, whereas the clinical sites prioritized more research variants/candidate genes (P < .001). The clinical sites solely identified 15% of the diagnoses/candidate genes. The differences between the 2 pipelines were more often because of variant prioritization disparities than variant detection.ConclusionThe unique dual analysis of NGS data in the UDN synergistically enhances outcomes. The core laboratory provided a clinical analysis with more diagnoses and the clinical sites prioritized more research variants/candidate genes. Implementing such concurrent dual analyses in other genomic research studies and clinical settings can improve both variant detection and prioritization.     

Sclerosing TSC1 mutated renal cell carcinoma: An unusual pattern mimicking MITF family translocation renal cell carcinoma

The tuberous sclerosis genes and MTOR are increasingly being found to have important roles in novel subtypes of renal cancer, particularly emerging entities eosinophilic solid and cystic renal cell carcinoma (RCC) and high‐grade oncocytic renal tumor (HOT)/RCC with eosinophilic and vacuolated cytoplasm. We report a unique renal neoplasm in a 66‐year‐old woman that initially mimicked MITF family translocation RCC due to mixed clear and eosinophilic cells, extensive stromal hyalinization, and psammoma bodies, yet which was negative for TFE3 and TFEB fluorescence in situ hybridization and a next generation sequencing (NGS) gene fusion assay. Cytoplasmic stippling triggered consideration of TSC‐associated neoplasms, and a targeted NGS assay revealed a variant in exon 21 of TSC1 resulting in c.2626G>T p.(Glu876*) truncating mutation. This report adds to the morphologic spectrum of TSC‐related renal neoplasms, including prominent stromal hyalinization as a potentially deceptive pattern. Due to the overlap in cytoplasmic stippling between eosinophilic solid and cystic RCC and HOT/RCC with eosinophilic and vacuolated cytoplasm, it is debatable which category this example would best fit. Further understanding of these entities and other renal neoplasms with alterations in the TSC genes will elucidate whether they should be considered a family of tumors.     

Targeted next-generation sequencing makes new molecular diagnoses and expands genotype–phenotype relationship in Ehlers–Danlos syndrome

PurposeEhlers–Danlos syndrome (EDS) comprises a group of overlapping hereditary disorders of connective tissue with significant morbidity and mortality, including major vascular complications. We sought to identify the diagnostic utility of a next-generation sequencing (NGS) panel in a mixed EDS cohort.MethodsWe developed and applied PCR-based NGS assays for targeted, unbiased sequencing of 12 collagen and aortopathy genes to a cohort of 177 unrelated EDS patients. Variants were scored blind to previous genetic testing and then compared with results of previous Sanger sequencing.ResultsTwenty-eight pathogenic variants in COL5A1/2, COL3A1, FBN1, and COL1A1 and four likely pathogenic variants in COL1A1, TGFBR1/2, and SMAD3 were identified by the NGS assays. These included all previously detected single-nucleotide and other short pathogenic variants in these genes, and seven newly detected pathogenic or likely pathogenic variants leading to clinically significant diagnostic revisions. Twenty-two variants of uncertain significance were identified, seven of which were in aortopathy genes and required clinical follow-up.ConclusionUnbiased NGS-based sequencing made new molecular diagnoses outside the expected EDS genotype–phenotype relationship and identified previously undetected clinically actionable variants in aortopathy susceptibility genes. These data may be of value in guiding future clinical pathways for genetic diagnosis in EDS.Genet Med 18 11, 1119–1127.     

Results of next‐generation sequencing gene panel diagnostics including copy‐number variation analysis in 810 patients suspected of heritable thoracic aortic disorders

Simultaneous analysis of multiple genes using next‐generation sequencing (NGS) technology has become widely available. Copy‐number variations (CNVs) in disease‐associated genes have emerged as a cause for several hereditary disorders. CNVs are, however, not routinely detected using NGS analysis. The aim of this study was to assess the diagnostic yield and the prevalence of CNVs using our panel of Hereditary Thoracic Aortic Disease (H‐TAD)‐associated genes. Eight hundred ten patients suspected of H‐TAD were analyzed by targeted NGS analysis of 21 H‐TAD associated genes. In addition, the eXome hidden Markov model (XHMM; an algorithm to identify CNVs in targeted NGS data) was used to detect CNVs in these genes. A pathogenic or likely pathogenic variant was found in 66 of 810 patients (8.1%). Of these 66 pathogenic or likely pathogenic variants, six (9.1%) were CNVs not detectable by routine NGS analysis. These CNVs were four intragenic (multi‐)exon deletions in MYLK, TGFB2, SMAD3, and PRKG1, respectively. In addition, a large duplication including NOTCH1 and a large deletion encompassing SCARF2 were detected. As confirmed by additional analyses, both CNVs indicated larger chromosomal abnormalities, which could explain the phenotype in both patients. Given the clinical relevance of the identification of a genetic cause, CNV analysis using a method such as XHMM should be incorporated into the clinical diagnostic care for H‐TAD patients.