Congenital defects of the erythrocyte membrane are common in northern Europe and all over the world. The resulting diseases, for example, hereditary spherocytosis (HS), are often underdiagnosed, partly due to their sometimes mild and asymptomatic courses. In addition to a broad clinical spectrum, this is also due to the occasionally complex diagnostics that are not available to every patient. To test whether next-generation sequencing (NGS) could replace time-consuming spherocytosis-specific functional tests, 22 consecutive patients with suspected red cell membranopathy underwent functional blood tests. We were able to identify the causative genetic defect in all patients with suspected HS who underwent genetic testing (n = 17). The sensitivity of the NGS approach, which tests five genes (ANK1 (gene product: ankyrin1), EPB42 (erythrocyte membrane protein band4.2), SLC4A1 (band3), SPTA1 (α-spectrin), and SPTB (β-spectrin)), was 100% (95% confidence interval: 81.5–100.0%). The major advantage of genetic testing in the paediatric setting is the small amount of blood required (<200 µL), and compared to functional assays, sample stability is not an issue. The combination of medical history, basic laboratory parameters, and an NGS panel with five genes is sufficient for diagnosis in most cases. Only in rare cases, a more comprehensive functional screening is required.

Defects in primary or motile cilia result in a variety of human pathologies, and retinal degeneration is frequently associated with these so-called ciliopathies. We found that homozygosity for a truncating variant in CEP162, a centrosome and microtubule-associated protein required for transition zone assembly during ciliogenesis and neuronal differentiation in the retina, caused late-onset retinitis pigmentosa in 2 unrelated families. The mutant CEP162-E646R*5 protein was expressed and properly localized to the mitotic spindle, but it was missing from the basal body in primary and photoreceptor cilia. This impaired recruitment of transition zone components to the basal body and corresponded to complete loss of CEP162 function at the ciliary compartment, reflected by delayed formation of dysmorphic cilia. In contrast, shRNA knockdown of Cep162 in the developing mouse retina increased cell death, which was rescued by expression of CEP162-E646R*5, indicating that the mutant retains its role for retinal neurogenesis. Human retinal degeneration thus resulted from specific loss of the ciliary function of CEP162.

Germline defects in the transcription factor GATA1 are known to cause dyserythropoiesis with(out) anemia and variable abnormalities in platelet count and function. However, damaging variants closely located to the C-terminal zinc finger domain of GATA1 are nearly unknown. In this study, a 36-year-old male index patient and his 4-year-old daughter suffered from moderate mucocutaneous bleeding diathesis since birth. Whole exome sequencing detected a novel hemizygous GATA1 missense variant, c.886A>C p.T296P, located between the C-terminal zinc finger and the nuclear localization sequence with non-random X-chromosome inactivation in the heterozygous daughter. Blood smears from both patients demonstrated large platelet fractions and moderate thrombocytopenia in the index. Flow cytometry and electron microscopy analysis supported a combined α-/δ (AN-subtype)-storage pool deficiency as cause for impaired agonist-induced platelet aggregation (light transmission aggregometry) and granule exocytosis (flow cytometry). The absence of BCAM in the index (Lu(a-b-)) and its low expression in the daughter (Lu(a-b+)) confirmed a less obvious effect of defective GATA1 also on erythrocytes. Borderline anemia, elevated HbF levels, and differential transcription of GATA1-regulated genes indicated mild dyserythropoiesis in both patients. Furthermore, a mild SLC4A1 defect associated with a heterozygous SLC4A1 c.2210C>T p.A737V variant maternally transmitted in the daughter may modify the disease to mild spherocytosis and hemolysis.

MAD2L1BP -encoded p31 ^comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31 ^comet mRNA and protein. Corresponding recombinant p31 ^comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31 ^comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp ^–/– mice. Thus, human mutations of p31 ^comet are linked to aneuploidy and tumor predisposition.

Background: Severe high molecular weight kininogen (HK) deficiency is an autosomal recessive defect of the contact system caused by mutations in KNG1. Limited scientific interest in HK deficiency due to the rarity of the seemingly asymptomatic condition may increase, as HK, the precursor of bradykinin, is now discussed as a therapeutic target e.g. in hereditary angioedema. Aims: We provide a comprehensive analysis of the diagnostic, clinical, and genetic features of HK deficiency and estimate its frequency. Methods: We identified a new case of HK deficiency, systematically review the literature, conduct new genetic studies of reported cases, and comprehensively analyze the clinical course and diagnostic criteria. Clotting activity of HK and prekallikrein (PK) (HK:C/PK:C) and antigen (HK:Ag/PK:Ag) were determined and genetic analyses of KNG1 and KLKB1 were performed by Sanger sequencing. Characteristics deduced from the known HK deficiency-causing variants were used to estimate the frequency of HK deficiency from the KNG1 variants aggregated in GnomAD. Results: 677 studies were identified by systematic review of the literature for HK deficiency. 27 of these contained individual cases of HK deficiency including 6 studies not listed in PubMed. Little-noticed cases from the gray literature account for more than one-third (16/39) of the extracted, unrelated cases. We genotyped one newly diagnosed HK-deficient case and 2 cases described in the literature and additionally evaluated all 10 studies reporting genetic data in HK-deficiency (including one case previously misdiagnosed as having PK deficiency). A total of 10 KNG1 variants causing HK deficiency (one new) were found, the most frequent being c.586C>T, p.Arg196* (4 unrelated families). Interestingly, all HK deficiency-causing variants are truncating, whereas two amino acid substitutions with presumed functional consequence, have been described as the cause of hereditary angioedema. Conservative prevalence estimates based on all known and putative HK deficiency-causing variants extracted from GnomAD (truncating variants in KNG1, including indels, nonsense and canonical splice site mutations located in that part of the gene, where relevant mutations have been described) revealed a frequency of 1 case of HK deficiency among 7,925,172 with slight differences in the analyzed ethnicities (see table). In addition, although not to the same extent as seen in PK deficiency, HK deficiency apparently is more prevalent in Africans. While it is already well known that HK deficiency causes decreased PK levels, our data indicate that factor XI levels are also frequently decreased, albeit to a lesser extent. The number of cases detected so far is too low for a more detailed analysis regarding bleeding, thrombotic, and cardiovascular events or immunological abnormalities. Conclusion: HK-deficiency is probably more frequent than previously thought. Suspected cases of contact phase defects should at least be analyzed for HK activity (besides factor XII, XI and PK activity) to facilitate conclusive evaluation of the clinical significance in the future.