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The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as “maybe report” after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

An increasing number of individuals with intellectual developmental disorder (IDD) and heterozygous variants in BCL11Aare identified, yet our knowledge of manifestations and mutational spectrum is lacking. To address this, we performed detailed analysis of 42 individuals with BCL11A-related IDD (BCL11A-IDD, a.k.a. Dias-Logan syndrome) ascertained through an international collaborative network, and reviewed 35 additional previously reported patients. Analysis of 77 affected individuals identified 60 unique disease-causing variants (30 frameshift, 7 missense, 6 splice-site, 17 stop-gain) and 8 unique BCL11Amicrodeletions. We define the most prevalent features of BCL11A-IDD: IDD, postnatal-onset microcephaly, hypotonia, behavioral abnormalities, autism spectrum disorder, and persistence of fetal hemoglobin (HbF), and identify autonomic dysregulation as new feature. BCL11A-IDD is distinguished from 2p16 microdeletion syndrome, which has a higher incidence of congenital anomalies. Our results underscore BCL11A as an important transcription factor in human hindbrain development, identifying a previously underrecognized phenotype of a small brainstem with a reduced pons/medulla ratio. Genotype-phenotype correlation revealed an isoform-dependent trend in severity of truncating variants: those affecting all isoforms are associated with higher frequency of hypotonia, and those affecting the long (BCL11A-L) and extra-long (-XL) isoforms, sparing the short (-S), are associated with higher frequency of postnatal microcephaly. With the largest international cohort to date, this study highlights persistence of fetal hemoglobin as a consistent biomarker and hindbrain abnormalities as a common feature. It contributes significantly to our understanding of BCL11A-IDD through an extensive unbiased multi-center assessment, providing valuable insights for diagnosis, management and counselling, and into BCL11A’s role in brain development.

The American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) system for variant classification is score based with five classes: benign, likely benign, variant of unknown significance (VUS), likely pathogenic, and pathogenic. Here, we present a variant classification model that can be an add-on or alternative to ACMG classification: A stepwise system that can classify any type of genetic variant (e.g., hypomorphic alleles, imprinted alleles, copy number variants, runs of homozygosity, enhancer variants, and variants related to traits). We call it the ABC system because classification is first functional (A), then clinical (B), and optionally a standard comment that fits the clinical question is selected (C). Both steps A and B have 1–5 grading when knowledge is sufficient, if not, class “zero” is assigned. Functional grading (A) only concerns biological consequences with the stages normal function (1), likely normal function (2), hypothetical functional effect (3), likely functional effect (4), and proven functional effect (5). Clinical grading (B) is genotype–phenotype focused with the stages “right type of gene” (1), risk factor (2), and pathogenic (3–5, depending on penetrance). Both grades are listed for each variant and combined to generate a joint class ranging from A to F. Importantly, the A–F classes are linked to standard comments, reflecting laboratory or national policy. In step A, the VUS class is split into class 0 (true unknown) and class 3 (hypothetical functional effect based on molecular predictions or de novo occurrence), providing a rationale for variant-of-interest reporting when the clinical picture could fit the finding. The system gives clinicians a better guide to variant significance.

In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.

Recurrent copy number variations (CNVs) are common causes of neurodevelopmental disorders (NDDs) and associated with a range of psychiatric traits. These CNVs occur at defined genomic regions that are particularly prone to recurrent deletions and duplications and often exhibit variable expressivity and incomplete penetrance. Robust estimates of the population prevalence and inheritance pattern of recurrent CNVs associated with neurodevelopmental disorders (NDD CNVs) are lacking. Here we perform array-based CNV calling in 12,252 mother–father–child trios from the Norwegian Mother, Father, and Child Cohort Study (MoBa) and analyse the inheritance pattern of 26 recurrent NDD CNVs in 13 genomic regions. We estimate the total prevalence of recurrent NDD CNVs (duplications and deletions) in live-born children to 0.48% (95% C.I.: 0.37–0.62%), i.e., ~1 in 200 newborns has either a deletion or duplication in these NDDs associated regions. Approximately a third of the newborn recurrent NDD CNVs (34%, N?=?20/59) are de novo variants. We provide prevalence estimates and inheritance information for each of the 26 NDD CNVs and find higher prevalence than previously reported for 1q21.1 deletions (~1:2000), 15q11.2 duplications (~1:4000), 15q13.3 microdeletions (~1:2500), 16p11.2 proximal microdeletions (~1:2000) and 17q12 deletions (~1:4000) and lower than previously reported prevalence for the 22q11.2 deletion (~1:12,000). In conclusion, our analysis of an unselected and representative population of newborns and their parents provides a clearer picture of the rate of recurrent microdeletions/duplications implicated in neurodevelopmental delay. These results will provide an important resource for genetic diagnostics and counseling.

Rare sequence variants in the non-coding part of the BRCAgenes are often reported as variants of uncertain significance (VUS), which leave patients and doctors in a challenging position. The aim of this study was to determine the pathogenicity of the BRCA1c.5407-25T>A variant found in 20 families from Norway, France and United States with suspected hereditary breast and ovarian cancer. This was done by combining clinical and family information with allele frequency data, and assessment of the variant’s effect on mRNA splicing. Mean age at breast (n= 12) and ovarian (n= 11) cancer diagnosis in female carriers was 49.9 and 60.4 years, respectively. The mean Manchester score in the 20 families was 16.4. The allele frequency of BRCA1c.5407-25T>A was 1/64,566 in non-Finnish Europeans (gnomAD database v2.1.1). We found the variant in 1/400 anonymous Norwegian blood donors and 0/784 in-house exomes. Sequencing of patient-derived cDNA from blood, normal breast and ovarian tissue showed that BRCA1c.5407-25T>A leads to skipping of exon 23, resulting in frameshift and protein truncation: p.(Gly1803GlnfsTer11). Western blot analysis of transiently expressed BRCA1 proteins in HeLa cells showed a reduced amount of the truncated protein compared with wild type. Noteworthily, we found that a small amount of full-length transcript was also generated from the c.5407-25T>A allele, potentially explaining the intermediate cancer burden in families carrying this variant. In summary, our results show that BRCA1c.5407-25T>A leads to partial skipping of exon 23, and could represent a likely pathogenic variant with reduced penetrance.

Missense variants located to the “molecular brake” in the tyrosine kinase hinge region of platelet-derived growth factor receptor-β, encoded by PFGFRB, can cause Penttinen-type (Val665Ala) and Penttinen-like (Asn666His) premature ageing syndromes, as well as infantile myofibromatosis (Asn666Lys and Pro660Thr). We have found the same de novo PDGFRBc.1997A>G p.(Asn666Ser) variants in two patients with lipodystrophy, acro-osteolysis and severely reduced vision due to corneal neovascularisation, reminiscent of a severe form of Penttinen syndrome with more pronounced connective tissue destruction. In line with this phenotype, patient skin fibroblasts were prone to apoptosis. Both in patient fibroblasts and stably transduced HeLa and HEK293 cells, autophosphorylation of PDGFRβ was observed, as well as increased phosphorylation of downstream signalling proteins such as STAT1, PLCγ1, PTPN11/SHP2-Tyr580 and AKT. Phosphorylation of MAPK3 (ERK1) and PTPN11/SHP2-Tyr542 appeared unaffected. This suggests that this missense change not only weakens tyrosine kinase autoinhibition, but also influences substrate binding, as both PTPN11 tyrosines (Tyr542 and Tyr580) usually are phosphorylated upon PDGFR activation. Imatinib was a strong inhibitor of phosphorylation of all these targets, suggesting an option for precision medicine based treatment.

Potocki–Shaffer syndrome (PSS) is a contiguous gene syndrome caused by 11p11.2 deletions. PSS is clinically characterized by intellectual disability, craniofacial anomalies, enlarged parietal foramina, and multiple exostoses. PSS occasionally shows autism spectrum disorder, epilepsy, and overgrowth. Some of the clinical features are thought to be associated with haploinsufficiency of two genes in the 11p11.2 region; variants affecting the function of ALX4cause enlarged parietal foramina and EXT2lead to multiple exostoses. However, the remaining clinical features were still yet to be linked to specific genetic alterations. In this study, we identified de novo truncating variants in an 11p11.2 gene, PHF21A, in three cases with intellectual disability and craniofacial anomalies. Among these three cases, autism spectrum disorder was recognized in one case, epilepsy in one case, and overgrowth in two cases. This study shows that PHF21Ahaploinsufficiency results in intellectual disability and craniofacial anomalies and possibly contributes to susceptibility to autism spectrum disorder, epilepsy, and overgrowth, all of which are PSS features.

Whole-gene duplications and missense variants in the HUWE1gene (NM_031407.6) have been reported in association with intellectual disability (ID). Increased gene dosage has been observed in males with non-syndromic mild to moderate ID with speech delay. Missense variants reported previously appear to be associated with severe ID in males and mild or no ID in obligate carrier females. Here, we report the largest cohort of patients with HUWE1variants, consisting of 14 females and 7 males, with 15 different missense variants and one splice site variant. Clinical assessment identified common clinical features consisting of moderate to profound ID, delayed or absent speech, short stature with small hands and feet and facial dysmorphism consisting of a broad nasal tip, deep set eyes, epicanthic folds, short palpebral fissures, and a short philtrum. We describe for the first time that females can be severely affected, despite preferential inactivation of the affected X chromosome. Three females with the c.329 G  >  A p.Arg110Gln variant, present with a phenotype of mild ID, specific facial features, scoliosis and craniosynostosis, as reported previously in a single patient. In these females, the X inactivation pattern appeared skewed in favour of the affected transcript. In summary, HUWE1missense variants may cause syndromic ID in both males and females.

Heterozygous variants in BICD cargo adapter 2 (BICD2) cause autosomal dominant spinal muscular atrophy, lower extremity-predominant 2 (SMALED2). The disease is usually characterized by a benign or slowly progressive, congenital or early onset muscle weakness and atrophy that mainly affects the lower extremities, although some affected individuals show involvement of the arms and the shoulder girdle. Here we report unusual extremes of BICD2-related diseases: A severe form of congenital muscular atrophy with arthrogryposis multiplex, respiratory insufficiency and lethality within four months. This was caused by three BICD2 variants, (c.581A>G, p.(Gln194Arg)), (c.1626C>G, p.(Cys542Trp)) and (c.2080C>T, p.(Arg694Cys)), two of which were proven to be de novo. Affected individuals showed reduced fetal movement, weak muscle tone and sparse or no spontaneous activity after birth. Despite assisted ventilation, the condition led to early death. At the other extreme, we identified an asymptomatic woman with a known BICD2 variant (c.2108C>T, p.(Thr703Met)). Radiological examination showed fatty degeneration of selected thigh and calf muscles without clinical consequences. Instead, her son carrying the same variant is affected by a mild childhood onset disease with myopathic and neurogenic features. Mechanisms leading to variable expressivity and onset of BICD2-related disease may include alterations in molecular interactions of BICD2 and suggest the presence of genetic modifiers that may act in a protective fashion to ameliorate or abrogate disease. Our data define an additional severe disease type caused by BICD2 and emphasize a possibly variable etiology of BICD2-opathies with regard to primary muscle and neuronal involvement.