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Lynch Syndrome (LS) is a hereditary cancer syndrome caused by pathogenic germline variants in one of the four mismatch repair (MMR) genes MLH1 , MSH2 , MSH6 and PMS2 . It is characterized by a significantly increased risk of multiple cancer types, particularly colorectal and endometrial cancer, with autosomal dominant inheritance. Access to precise and sensitive methods for genetic testing is important, as early detection and prevention of cancer is possible when the variant is known. We present here two unrelated Norwegian families with family histories strongly suggestive of LS, where immunohistochemical and microsatellite instability analyses indicated presence of a pathogenic variant in MSH2 , but targeted exon sequencing and multiplex ligation-dependent probe amplification (MLPA) were negative. Using Bionano optical genome mapping, we detected a 39 kb insertion in the MSH2 gene. Precise mapping of the insertion breakpoints and inserted sequence was performed by low-coverage whole-genome sequencing with an Oxford Nanopore MinION. The same variant was present in both families, and later found in other families from the same region of Norway, indicative of a founder event. To our knowledge, this is the first diagnosis of LS caused by a structural variant using these technologies. We suggest that structural variant detection be performed when LS is suspected but not confirmed with first-tier standard genetic testing.

Purpose GATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype–phenotype correlations makes the decision regarding “who and when” to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT. Methods All medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients’ medical records. Results Between 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype–phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively. Conclusion Our main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the “optimal window” for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described.

Kinesin proteins are critical for various cellular functions such as intracellular transport and cell division, and many members of the family have been linked to monogenic disorders and cancer. We report eight individuals with intellectual disability and microcephaly from four unrelated families with parental consanguinity. In the affected individuals of each family, homozygosity for likely pathogenic variants in KIF14 were detected; two loss-of-function (p.Asn83Ilefs*3 and p.Ser1478fs), and two missense substitutions (p.Ser841Phe and p.Gly459Arg). KIF14 is a mitotic motor protein that is required for spindle localization of the mitotic citron rho-interacting kinase, CIT, also mutated in microcephaly. Our results demonstrate the involvement of KIF14 in development and reveal a wide phenotypic variability ranging from fetal lethality to moderate developmental delay and microcephaly.

Severe combined immunodeficiency (SCID) and other T cell lymphopenias can be detected during newborn screening (NBS) by measuring T cell receptor excision circles (TRECs) in dried blood spot (DBS) DNA. Second tier next generation sequencing (NGS) with an amplicon based targeted gene panel using the same DBS DNA was introduced as part of our prospective pilot research project in 2015. With parental consent, 21 000 newborns were TREC-tested in the pilot. Three newborns were identified with SCID, and disease-causing variants in , and were confirmed by NGS on the initial DBS DNA. The molecular findings directed follow-up and therapy: the -SCID underwent early hematopoietic stem cell transplantation (HSCT) without any complications; the leaky SCID received prophylactic antibiotics, antifungals, and immunoglobulin infusions, and underwent HSCT at 1 year of age. The child with -SCID had complete Hirschsprung disease and died at 1 month of age. Since January 2018, all newborns in Norway have been offered NBS for SCID using 1st tier TRECs and 2nd tier gene panel NGS on DBS DNA. During the first 20 months of nationwide SCID screening an additional 88 000 newborns were TREC tested, and four new SCID cases were identified. Disease-causing variants in , and were molecularly confirmed on day 8, 15, 8 and 6, respectively after birth, using the initial NBS blood spot. Targeted gene panel NGS integrated into the NBS algorithm rapidly delineated the specific molecular diagnoses and provided information useful for management, targeted therapy and follow-up i.e., X rays and CT scans were avoided in the radiosensitive SCID. Second tier targeted NGS on the same DBS DNA as the TREC test provided instant confirmation or exclusion of SCID, and made it possible to use a less stringent TREC cut-off value. This allowed for the detection of leaky SCIDs, and simultaneously reduced the number of control samples, recalls and false positives. Mothers were instructed to stop breastfeeding until maternal (CMV) status was determined. Our limited data suggest that shorter time-interval from birth to intervention, may prevent breast milk transmitted CMV infection in classical SCID.

Background Four patients from three Norwegian families presented with a common skin phenotype of warts, molluscum contagiosum, and dermatitis since early childhood, and various other immunological features. Warts are a common manifestation of human papilloma virus (HPV), but when they are overwhelming, disseminated and/or persistent, and presenting together with other immunological features, a primary immunodeficiency disease (PIDD) may be suspected. Methods and results The four patients were exome sequenced as part of a larger study for detecting genetic causes of primary immunodeficiencies. No disease‐causing variants were identified in known primary immunodeficiency genes or in other disease‐related OMIM genes. However, the same homozygous missense variant in CARMIL2 (also known as RLTPR) was identified in all four patients. In each family, the variant was located within a narrow region of homozygosity, representing a potential region of autozygosity. CARMIL2 is a protein of undetermined function. A role in T‐cell activation has been suggested and the mouse protein homolog (Rltpr) is essential for costimulation of T‐cell activation via CD28, and for the development of regulatory T cells. Immunophenotyping demonstrated reduced regulatory, CD4+ memory, and CD4+ follicular T cells in all four patients. In addition, they all seem to have a deficiency in IFNγ ‐synthesis in CD4+ T cells and NK cells. Conclusions We report a novel primary immunodeficiency, and a differential molecular diagnosis to CXCR4‐, DOCK8‐, GATA2‐, MAGT1‐, MCM4‐, STK4‐, RHOH‐, TMC6‐, and TMC8‐related diseases. The specific variant may represent a Norwegian founder variant segregating on a population‐specific haplotype. A likely Norwegian founder variant was detected by exome sequencing in four patients from three different families. The patients had signs of primary immunodeficiency and common skin phenotype of warts, molluscs, and dermatitis since early childhood.

Background With advances in next generation sequencing technologies and genomic capture techniques, exome sequencing has become a cost-effective approach for mutation detection in genetic diseases. However, computational prediction of copy number variants (CNVs) from exome sequence data is a challenging task. Whilst numerous programs are available, they have different sensitivities, and have low sensitivity to detect smaller CNVs (1–4 exons). Additionally, exonic CNV discovery using standard aCGH has limitations due to the low probe density over exonic regions. The goal of our study was to develop a protocol to detect exonic CNVs (including shorter CNVs that cover 1–4 exons), combining computational prediction algorithms and a high-resolution custom CGH array. Results We used six published CNV prediction programs (ExomeCNV, CONTRA, ExomeCopy, ExomeDepth, CoNIFER, XHMM) and an in-house modification to ExomeCopy and ExomeDepth (ExCopyDepth) for computational CNV prediction on 30 exomes from the 1000 genomes project and 9 exomes from primary immunodeficiency patients. CNV predictions were tested using a custom CGH array designed to capture all exons (exaCGH). After this validation, we next evaluated the computational prediction of shorter CNVs. ExomeCopy and the in-house modified algorithm, ExCopyDepth, showed the highest capability in detecting shorter CNVs. Finally, the performance of each computational program was assessed by calculating the sensitivity and false positive rate. Conclusions In this paper, we assessed the ability of 6 computational programs to predict CNVs, focussing on short (1–4 exon) CNVs. We also tested these predictions using a custom array targeting exons. Based on these results, we propose a protocol to identify and confirm shorter exonic CNVs combining computational prediction algorithms and custom aCGH experiments.

We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.

Six kindreds had heterozygous mutations in the gene for the IKAROS transcription factor. Twenty-nine patients had late-onset hypogammaglobulinemia, a decrease in peripheral-blood B cells, and normal memory B cells and plasma cells. Acute lymphoblastic leukemia developed in two patients. The genetic cause of many primary immunodeficiencies remains unknown. Common variable immunodeficiency (CVID) comprises a heterogeneous group of disorders characterized by the late onset of recurrent infections, hypogammaglobulinemia, and poor antibody response to vaccine antigens that cannot be explained by previous exposures, treatment, or infections. Some patients also have autoimmunity, granulomatous disease, or cancer. 1 , 2 Genomic approaches, including whole-exome sequencing and high-resolution array-based comparative genomic hybridization (CGH), have accelerated the identification of genetic causes in patients with primary immunodeficiencies, including CVID. 3 – 5 Mutations in several genes, including ICOS, CD19, CD81, CD20, CD21, TWEAK, CTLA4, LRBA, GATA2, CXCL12, NFKB1, and NFKB2, . . .

Background With advances in next generation sequencing technology and analysis methods, single nucleotide variants (SNVs) and indels can be detected with high sensitivity and specificity in exome sequencing data. Recent studies have demonstrated the ability to detect disease-causing copy number variants (CNVs) in exome sequencing data. However, exonic CNV prediction programs have shown high false positive CNV counts, which is the major limiting factor for the applicability of these programs in clinical studies. Results We have developed a tool (cnvScan) to improve the clinical utility of computational CNV prediction in exome data. cnvScan can accept input from any CNV prediction program. cnvScan consists of two steps: CNV screening and CNV annotation. CNV screening evaluates CNV prediction using quality scores and refines this using an in-house CNV database, which greatly reduces the false positive rate. The annotation step provides functionally and clinically relevant information using multiple source datasets. We assessed the performance of cnvScan on CNV predictions from five different prediction programs using 64 exomes from Primary Immunodeficiency (PIDD) patients, and identified PIDD-causing CNVs in three individuals from two different families. Conclusions In summary, cnvScan reduces the time and effort required to detect disease-causing CNVs by reducing the false positive count and providing annotation. This improves the clinical utility of CNV detection in exome data.