Future of Dutch NGS-Based Newborn Screening: Exploring the Technical Possibilities and Assessment of a Variant Classification Strategy.

In this study, we compare next-generation sequencing (NGS) approaches (targeted panel (tNGS), whole exome sequencing (WES), and whole genome sequencing (WGS)) for application in newborn screening (NBS). DNA was extracted from dried blood spots (DBS) from 50 patients with genetically confirmed inherited metabolic disorders (IMDs) and 50 control samples. One hundred IMD-related genes were analyzed. Two data-filtering strategies were applied: one to detect only (likely) pathogenic ((L)P) variants, and one to detect (L)P variants in combination with variants of unknown significance (VUS). The variants were filtered and interpreted, defining true/false positives (TP/FP) and true/false negatives (TN/FN). The variant filtering strategies were assessed in a background cohort (BC) of 4833 individuals. Reliable results were obtained within 5 days. TP results (47 patient samples) for tNGS, WES, and WGS results were 33, 31, and 30, respectively, using the (L)P filtering, and 40, 40, and 38, respectively, when including VUS. FN results were 11, 13, and 14, respectively, excluding VUS, and 4, 4, and 6, when including VUS. The remaining FN were mainly samples with a homozygous VUS. All controls were TN. Three BC individuals showed a homozygous (L)P variant, all related to a variable, mild phenotype. The use of NGS-based workflows in NBS seems promising, although more knowledge of data handling, automated variant interpretation, and costs is needed before implementation.

A Delphi Survey Study to Formulate Statements on the Treatability of Inherited Metabolic Disorders to Decide on Eligibility for Newborn Screening.

The Wilson and Jungner (W&J) and Andermann criteria are meant to help select diseases eligible for population-based screening. With the introduction of next-generation sequencing (NGS) methods for newborn screening (NBS), more inherited metabolic diseases (IMDs) can technically be included, and a revision of the criteria was attempted. This study aimed to formulate statements and investigate whether those statements could elaborate on the criterion of treatability for IMDs to decide on eligibility for NBS. An online Delphi study was started among a panel of Dutch IMD experts (EPs). EPs evaluated, amended, and approved statements on treatability that were subsequently applied to 10 IMDs. After two rounds of Delphi, consensus was reached on 10 statements. Application of these statements selected 5 out of 10 IMDs proposed for this study as eligible for NBS, including 3 IMDs in the current Dutch NBS. The statement: 'The expected benefit/burden ratio of early treatment is positive and results in a significant health outcome' contributed most to decision-making. Our Delphi study resulted in 10 statements that can help to decide on eligibility for inclusion in NBS based on treatability, also showing that other criteria could be handled in a comparable way. Validation of the statements is required before these can be applied as guidance to authorities.

Lessons Learned from 17 Years of Multidisciplinary Care for DSD Patients at A Single Indonesian Center.

BACKGROUND: Our Multidisciplinary Team (MDT) is a large specialized team based in Semarang, Indonesia that cares for a wide variety of pediatric and adult individuals with Differences of Sex Development (DSD) from across Indonesia. Here we describe our work over the last 17 years. METHODS: We analyzed phenotypic, hormonal and genetic findings from clinical records for all patients referred to our MDT during the period 2004 to 2020. RESULTS: Among 1184 DSD patients, 10% had sex chromosome DSD, 67% had 46,XY DSD and 23% had 46,XX DSD. The most common sex chromosome anomaly was Turner syndrome (45,X) (55 cases). For patients with 46,XY DSD under-masculinization was the most common diagnosis (311 cases) and for 46,XX DSD a defect of Müllerian development was most common (131 cases) followed by Congenital Adrenal Hyperplasia (CAH) (116 cases). Sanger sequencing, MLPA and targeted gene sequencing of 257 patients with 46,XY DSD found likely causative variants in 21% (55 cases), with 13 diagnostic genes implicated. The most affected gene coded for the Androgen Receptor. Molecular analysis identified a diagnosis for 69 of 116 patients with CAH, with 62 carrying variants in CYP21A2 including four novel variants, and seven patients carrying variants in CYP11B1. In many cases these genetic diagnoses influenced the clinical management of patients and families. CONCLUSIONS: Our work has highlighted the occurrence of different DSDs in Indonesia. By applying sequencing technologies as part of our clinical care, we have delivered a number of genetic diagnoses and identified novel pathogenic variants in some genes, which may be clinically specific to Indonesia. Genetics can inform many aspects of DSD clinical management, and whilst many of our patients remain undiagnosed, we hope that future testing may provide answers for even more.

Novel likely pathogenic variant in NR5A1 gene in a Tanzanian child with 46,XY differences of sex development, inherited from the mosaic father.

Summary: Pathogenic variants in the nuclear receptor subfamily 5 group A member 1 gene (NR5A1), which encodes steroidogenic factor 1 (SF1), result in 46,XY and 46,XX differences of sex development (DSD). In 46,XY individuals with a pathogenic variant in the NR5A1 gene a variable phenotype ranging from mild to severe is seen, including adrenal failure, testis dysgenesis, androgen synthesis defects, hypospadias and anorchia with microphallus and infertility. We report the clinical, endocrinological and genetic characteristics of a patient with 46,XY DSD with a novel likely pathogenic missense variant in the NR5A1 gene. A retrospective evaluation of the medical history, physical examination, limited endocrinological laboratory analysis and genetic analysis with DSD gene panel testing was performed. A 1.5-month-old individual was referred with ambiguous genitalia. The karyotype was 46,XY. The endocrinological analyses were within normal male reference including a normal response of cortisol within an adrenocorticotropic hormone test. A novel heterozygous missense variant c.206G>C p.(Arg69Pro) in the NR5A1 gene was detected. This variant was present in mosaic form (~20%) in his unaffected father. Because another missense variant at the same position and other missense variants involving the same highly conserved codon have been reported, we consider this NR5A1 variant in this 46,XY DSD patient as likely pathogenic in accordance with the ACMG/AMP 2015 guidelines causing ambiguous genitalia but no adrenal insufficiency. This variant was inherited from the apparently unaffected mosaic father, which might have implications for the recurrence risk in this family. Learning points: The importance of performing trio (patient and parents) sequencing is crucial in pointing out the origin of inheritance. In a 46,XY differences of sex development patient, a normal adrenal function does not rule out an NR5A1 mutation. With the support of a dedicated overseas institute partnership, we could solve this complex clinical case by molecular diagnosis in a resource-limited setting.

All-in-one whole exome sequencing strategy with simultaneous copy number variant, single nucleotide variant and absence-of-heterozygosity analysis in fetuses with structural ultrasound anomalies: A 1-year experience.

OBJECTIVE: We performed a 1-year evaluation of a novel strategy of simultaneously analyzing single nucleotide variants (SNVs), copy number variants (CNVs) and copy-number-neutral Absence-of-Heterozygosity from Whole Exome Sequencing (WES) data for prenatal diagnosis of fetuses with ultrasound (US) anomalies and a non-causative QF-PCR result. METHODS: After invasive diagnostics, whole exome parent-offspring trio-sequencing with exome-wide CNV analysis was performed in pregnancies with fetal US anomalies and a non-causative QF-PCR result (WES-CNV). On request, additional SNV-analysis, restricted to (the) requested gene panel(s) only (with the option of whole exome SNV-analysis afterward) was performed simultaneously (WES-CNV/SNV) or as rapid SNV-re-analysis, following a normal CNV analysis. RESULTS: In total, 415 prenatal samples were included. Following a non-causative QF-PCR result, WES-CNV analysis was initially requested for 74.3% of the chorionic villus (CV) samples and 45% of the amniotic fluid (AF) samples. In case WES-CNV analysis did not reveal a causative aberration, SNV-re-analysis was requested in 41.7% of the CV samples and 17.5% of the AF samples. All initial analyses could be finished within 2 weeks after sampling. For SNV-re-analysis during pregnancy, turn-around-times (TATs) varied between one and 8 days. CONCLUSION: We show a highly efficient all-in-one WES-based strategy, with short TATs, and the option of rapid SNV-re-analysis after a normal CNV result.

Lessons learned from rapid exome sequencing for 575 critically ill patients across the broad spectrum of rare disease.

Introduction: Rapid exome sequencing (rES) has become the first-choice genetic test for critically ill patients, mostly neonates, young infants, or fetuses in prenatal care, in time-sensitive situations and when it is expected that the genetic test result may guide clinical decision making. The implementation of rES has revolutionized medicine by enabling timely identification of genetic causes for various rare diseases. The utilization of rES has increasingly been recognized as an essential diagnostic tool for the identification of complex and undiagnosed genetic disorders. Methods: We conducted a retrospective evaluation of our experiences with rES performed on 575 critically ill patients from various age groups (prenatal to adulthood), over a four-year period (2016-2019). These patients presented with a wide spectrum of rare diseases, including but not limited to neurological disorders, severe combined immune deficiency, and cancer. Results: During the study period, there was a significant increase in rES referrals, with a rise from a total of two referrals in Q1-2016 to 10 referrals per week in Q4-2019. The median turnaround time (TAT) decreased from 17 to 11 days in the period 2016-2019, with an overall median TAT of 11 days (IQR 8-15 days). The overall diagnostic yield for this cohort was 30.4%, and did not significantly differ between the different age groups (e.g. adults 22.2% vs children 31.0%; p-value 0.35). However, variability in yield was observed between clinical entities: craniofacial anomalies yielded 58.3%, while for three clinical entities (severe combined immune deficiency, aneurysm, and hypogonadotropic hypogonadism) no diagnoses were obtained. Discussion: Importantly, whereas clinical significance is often only attributed to a conclusive diagnosis, we also observed impact on clinical decision-making for individuals in whom no genetic diagnosis was established. Hence, our experience shows that rES has an important role for patients of all ages and across the broad spectrum of rare diseases to impact clinical outcomes.

The genetic diagnosis of rare endocrine disorders of sex development and maturation: a survey among Endo-ERN centres.

Differences of sex development and maturation (SDM) represent a heterogeneous puzzle of rare conditions with a large genetic component whose management and treatment could be improved by an accurate classification of underlying molecular conditions, and next-generation sequencing (NGS) should represent the most appropriate approach. Therefore, we conducted a survey dedicated to the use and potential outcomes of NGS for SDM disorders diagnosis among the 53 health care providers (HCP) of the European Reference Network for rare endocrine conditions. The response rate was 49% with a total of 26 HCPs from 13 countries. All HCPs, except 1, performed NGS investigations for SDM disorders on 6720 patients, 3764 (56%) with differences of sex development (DSD), including 811 unexplained primary ovarian insufficiency, and 2956 (44%) with congenital hypogonadotropic hypogonadism (CHH). The approaches varied from targeted analysis of custom gene panels (range: 11-490 genes) in 81.5% of cases or whole exome sequencing with the extraction of a virtual panel in the remaining cases. These analyses were performed for diagnostic purposes in 21 HCPs, supported by the National Health Systems in 16 cases. The likelihood of finding a variant ranged between 7 and 60%, mainly depending upon the number of analysed genes or criteria used for reporting, most HCPs also reporting variants of uncertain significance. These data illustrate the status of genetic diagnosis of DSD and CHH across Europe. In most countries, these analyses are performed for diagnostic purposes, yielding highly variable results, thus suggesting the need for harmonization and general improvements of NGS approaches.

Towards Next-Generation Sequencing (NGS)-Based Newborn Screening: A Technical Study to Prepare for the Challenges Ahead.

Newborn screening (NBS) aims to identify neonates with severe conditions for whom immediate treatment is required. Currently, a biochemistry-first approach is used to identify these disorders, which are predominantly inherited meta1bolic disorders (IMD). Next-generation sequencing (NGS) is expected to have some advantages over the current approach, for example the ability to detect IMDs that meet all screening criteria but lack an identifiable biochemical footprint. We have now designed a technical study to explore the use of NGS techniques as a first-tier approach in NBS. Here, we describe the aim and set-up of the NGS-first for the NBS (NGSf4NBS) project, which will proceed in three steps. In Step 1, we will identify IMDs eligible for NGS-first testing, based on treatability. In Step 2, we will investigate the feasibility, limitations and comparability of different technical NGS approaches and analysis workflows for NBS, eventually aiming to develop a rapid NGS-based workflow. Finally, in Step 3, we will prepare for the incorporation of this workflow into the existing Dutch NBS program and propose a protocol for referral of a child after a positive NGS test result. The results of this study will be the basis for an additional analytical route within NBS that will be further studied for its applicability within the NBS program, e.g., regarding the ethical, legal, financial and social implications.

Long-term follow-up including extensive complement analysis of a pediatric C3 glomerulopathy cohort.

BACKGROUND: C3 glomerulopathy (C3G) is a rare kidney disorder characterized by predominant glomerular depositions of complement C3. C3G can be subdivided into dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). This study describes the long-term follow-up with extensive complement analysis of 29 Dutch children with C3G. METHODS: Twenty-nine C3G patients (19 DDD, 10 C3GN) diagnosed between 1992 and 2014 were included. Clinical and laboratory findings were collected at presentation and during follow-up. Specialized assays were used to detect rare variants in complement genes and measure complement-directed autoantibodies and biomarkers in blood. RESULTS: DDD patients presented with lower estimated glomerular filtration rate (eGFR). C3 nephritic factors (C3NeFs) were detected in 20 patients and remained detectable over time despite immunosuppressive treatment. At presentation, low serum C3 levels were detected in 84% of all patients. During follow-up, in about 50% of patients, all of them C3NeF-positive, C3 levels remained low. Linear mixed model analysis showed that C3GN patients had higher soluble C5b-9 (sC5b-9) and lower properdin levels compared to DDD patients. With a median follow-up of 52 months, an overall benign outcome was observed with only six patients with eGFR below 90 ml/min/1.73 m2 at last follow-up. CONCLUSIONS: We extensively described clinical and laboratory findings including complement features of an exclusively pediatric C3G cohort. Outcome was relatively benign, persistent low C3 correlated with C3NeF presence, and C3GN was associated with higher sC5b-9 and lower properdin levels. Prospective studies are needed to further elucidate the pathogenic mechanisms underlying C3G and guide personalized medicine with complement therapeutics.

Panel-Based Exome Sequencing for Neuromuscular Disorders as a Diagnostic Service.

BACKGROUND: Neuromuscular disorders (NMDs) are clinically and genetically heterogeneous. Accurate molecular genetic diagnosis can improve clinical management, provides appropriate genetic counseling and testing of relatives, and allows potential therapeutic trials. OBJECTIVE: To establish the clinical utility of panel-based whole exome sequencing (WES) in NMDs in a population with children and adults with various neuromuscular symptoms. METHODS: Clinical exome sequencing, followed by diagnostic interpretation of variants in genes associated with NMDs, was performed in a cohort of 396 patients suspected of having a genetic cause with a variable age of onset, neuromuscular phenotype, and inheritance pattern. Many had previously undergone targeted gene testing without results. RESULTS: Disease-causing variants were identified in 75/396 patients (19%), with variants in the three COL6-genes (COL6A1, COL6A2 and COL6A3) as the most common cause of the identified muscle disorder, followed by variants in the RYR1 gene. Together, these four genes account for almost 25% of cases in whom a definite genetic cause was identified. Furthermore, likely pathogenic variants and/or variants of uncertain significance were identified in 95 of the patients (24%), in whom functional and/or segregation analysis should be used to confirm or reject the pathogenicity. In 18% of the cases with a disease-causing variant of which we received additional clinical information, we identified a genetic cause in genes of which the associated phenotypes did not match that of the patients. Hence, the advantage of panel-based WES is its unbiased approach. CONCLUSION: Whole exome sequencing, followed by filtering for NMD genes, offers an unbiased approach for the genetic diagnostics of NMD patients. This approach could be used as a first-tier test in neuromuscular disorders with a high suspicion of a genetic cause. With uncertain results, functional testing and segregation analysis are needed to complete the evidence.

Autosomal recessive limb-girdle and Miyoshi muscular dystrophies in the Netherlands: The clinical and molecular spectrum of 244 patients.

In this retrospective study, we conducted a clinico-genetic analysis of patients with autosomal recessive limb-girdle muscular dystrophy (LGMD) and Miyoshi muscular dystrophy (MMD). Patients were identified at the tertiary referral centre for DNA diagnosis in the Netherlands and included if they carried two mutations in CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TRIM32, FKRP or ANO5 gene. DNA was screened by direct sequencing and multiplex ligand-dependent probe amplification (MLPA) analysis. A total of 244 patients was identified; 68 LGMDR1/LGMD2A patients with CAPN3 mutations (28%), 67 sarcoglycanopathy patients (LGMDR3-5/LGMD2C-E) (27%), 64 LGMDR12/LGMD2L and MMD3 patients with ANO5 mutations (26%), 25 LGMDR2/LGMD2B and MMD1 with DYSF mutations (10%), 21 LGMDR9/LGMD2I with FKRP mutations (9%) and one LGMDR8/LGMD2H patient with TRIM32 mutations (<1%). The estimated minimum prevalence of AR-LGMD and MMD in the Netherlands amounted to 14.4 × 10-6 . Thirty-three novel mutations were identified. A wide range in age of onset (0-72 years) and loss of ambulation (5-74 years) was found. Fifteen patients (6%) initially presented with asymptomatic hyperCKemia. Cardiac abnormalities were found in 35 patients (17%). Non-invasive ventilation was started in 34 patients (14%). Both cardiac and respiratory involvement occurs across all subtypes, stressing the need for screening in all included subtypes.

Serological and genetic complement alterations in infection-induced and complement-mediated hemolytic uremic syndrome.

BACKGROUND: The role of complement in the atypical form of hemolytic uremic syndrome (aHUS) has been investigated extensively in recent years. As the HUS-associated bacteria Shiga-toxin-producing Escherichia coli (STEC) can evade the complement system, we hypothesized that complement dysregulation is also important in infection-induced HUS. METHODS: Serological profiles (C3, FH, FI, AP activity, C3d, C3bBbP, C3b/c, TCC, αFH) and genetic profiles (CFH, CFI, CD46, CFB, C3) of the alternative complement pathway were prospectively determined in the acute and convalescent phase of disease in children newly diagnosed with STEC-HUS or aHUS. Serological profiles were compared with those of 90 age-matched controls. RESULTS: Thirty-seven patients were studied (26 STEC-HUS, 11 aHUS). In 39 % of them, including 28 % of STEC-HUS patients, we identified a genetic and/or acquired complement abnormality. In all patient groups, the levels of investigated alternative pathway (AP) activation markers were elevated in the acute phase and normalized in remission. The levels were significantly higher in aHUS than in STEC-HUS patients. CONCLUSIONS: In both infection-induced HUS and aHUS patients, complement is activated in the acute phase of the disease but not during remission. The C3d/C3 ratio displayed the best discrepancy between acute and convalescent phase and between STEC-HUS and aHUS and might therefore be used as a biomarker in disease diagnosis and monitoring. The presence of aberrations in the alternative complement pathway in STEC-HUS patients was remarkable, as well.

Whole Exome Sequencing in Patients with the Cuticular Drusen Subtype of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in elderly people worldwide. Cuticular drusen (CD) is a clinical subtype of AMD, which typically displays an earlier age at onset, and has a strong genetic component. Genetic studies support a role for rare sequence variants in CD susceptibility, and rare sequence variants in the CFH gene have been identified in 8.8% of CD cases. To further explore the role of rare variants in CD, we performed whole exome sequencing (WES) in 14 affected members of six families and 12 sporadic cases with CD. We detected rare sequence variants in CFH and FBLN5, which previously were shown to harbor rare variants in patients with CD. In addition, we detected heterozygous rare sequence variants in several genes encoding components of the extracellular matrix (ECM), including FBLN1, FBLN3/EFEMP1, FBLN5, FBLN6/HMCN1, FBN2, and COL15A1. Two rare pathogenic variants were identified in the COL15A1 gene: one in a sporadic case and another was found to segregate in a family with six affected individuals with CD. In addition, two rare pathogenic variants were identified in the FGL1 gene in three unrelated CD cases. These findings suggest that alterations in the ECM and in the coagulation pathway may play a role in the pathogenesis of CD. The identified candidate genes require further analyses in larger cohorts to confirm their role in the CD subtype of AMD. No evidence was found of rare sequence variants in a single gene that segregate with CD in the six families, suggesting that the disease is genetically heterogeneous.

Complement Factor H Serum Levels Determine Resistance to Pneumococcal Invasive Disease.

Streptococcus pneumoniae is a major cause of life-threatening infections. Complement activation plays a vital role in opsonophagocytic killing of pneumococci in blood. Initial complement activation via the classical and lectin pathways is amplified through the alternative pathway amplification loop. Alternative pathway activity is inhibited by complement factor H (FH). Our study demonstrates the functional consequences of the variability in human serum FH levels on host defense. Using an in vivo mouse model combined with human in vitro assays, we show that the level of serum FH correlates with the efficacy of opsonophagocytic killing of pneumococci. In summary, we found that FH levels determine a delicate balance of alternative pathway activity, thus affecting the resistance to invasive pneumococcal disease. Our results suggest that variation in FH expression levels, naturally occurring in the human population, plays a thus far unrecognized role in the resistance to invasive pneumococcal disease.

[Partial lipodystrophy: a spot diagnosis]. / Partiële lipodystrofie: een diagnose à vue.

BACKGROUND: Partial lipodystrophy is a rare acquired disorder characterised by gradual loss of subcutaneous adipose tissue in the upper half of the body. CASE DESCRIPTION: We saw a 9-year-old girl who had been referred on account of recurrent urinary tract infections. On physical examination, she was noticed to be very thin in the face. Her upper extremities were also skinny. Strikingly, the lower half of her body was normally proportioned, which immediately suggested a diagnosis of partial lipodystrophy. Additional examinations showed a low level of complement factor C3 and the presence of C3 nephritic factor. CONCLUSION: Partial lipodystrophy is rare but it is important to include it in the differential diagnosis of unwanted disproportional subcutaneous fat loss because of the somatic and psychological consequences.

Sensitive, reliable and easy-performed laboratory monitoring of eculizumab therapy in atypical hemolytic uremic syndrome.

Complement C5 inhibitor eculizumab treatment in atypical hemolytic uremic syndrome is effective, but associated with high costs. Complement inhibition monitoring in these patients has not been standardized. In this study we evaluated novel functional assays for application in routine follow-up. We documented that the Wieslab® complement screen assay showed a sensitivity of 1-2% of C5 activity by adding purified C5 or normal human serum to a C5 deficient serum. All the patient samples obtained during the treatment course, were completely blocked for terminal complement pathway activity for up to four weeks after the eculizumab infusion. Levels of complexes between eculizumab and C5 were inversely correlated to the complement activity (p=0.01). Moreover, titrating serum from eculizumab-treated patients into normal serum revealed that eculizumab was present in excess up to four weeks after infusion. Thus, we demonstrate sensitive, reliable and easy-performed assays which can be used to design individual eculizumab dosage regimens.

Compound heterozygous mutations in the C6 gene of a child with recurrent infections.

The complement system plays an important role in both the innate and adaptive immune system. Patients with inherited complement deficiencies have an increased risk of systemic bacterial infections. Deficiencies of the terminal complement pathway are especially associated with invasive meningococcal disease. Here, we report a case of a boy that presented with arthritis and recurrent bacterial and viral infections. Extensive analyses revealed decreased complement activity of both classical and alternative pathway, indicating a deficiency of C3 or one of the factors of the terminal complement pathway. Mutational analysis of the C6 gene identified two compound heterozygous mutations. An unknown missense aberration was found that involves the loss of a cysteine, possibly affecting the 3D structure of the protein. Furthermore, a known splice site variation was identified that results in a 14% shorter protein, due to transcription of amino acids that are normally intronic until a stop codon is reached (exon-intron boundary defect). It is known that the protein with this latter aberration is still functionally active when present with other C6 mutations and therefore, the consequences of the combination of the identified variations have been studied. Quantitative ELISAs showed that at least one allele produced a circulating C6 molecule that can be incorporated in the membrane attack complex, likely the truncated protein. In the present case we observed relapsing bacterial and viral infections, but no meningococcal disease. The reduced complement activity can be explained by the identified genetic variations in C6, as recombinant C6 supplementation corrected complement function in vitro.

Severe infantile Bordetella pertussis pneumonia in monozygotic twins with a congenital C3 deficiency.

UNLABELLED: Bordetella pertussis or whooping cough is a vaccine-preventable disease that still remains a serious infection in neonates and young infants. We describe two young infants, monozygotic twins, with a severe B. pertussis pneumonia of whom one needed extracorporeal membrane oxygenation. Diagnostic work-up of unexplained hematuria and proteinuria during the illness revealed low serum complement component 3 (C3) levels. During follow-up, C3 levels remained low (400-600 mg/L). Extensive analysis of the persistent low C3 levels revealed an unknown heterozygous mutation in the C3 gene in both siblings and their mother. This C3 mutation in combination with the specific virulence mechanisms of B. pertussis probably contributed to the severe disease course in these cases. CONCLUSION: We propose that genetically caused complement disorders should be considered when confronted with severe cases of B. pertussis infection.

Combined pulmonary hypertension and renal thrombotic microangiopathy in cobalamin C deficiency.

Pulmonary arterial hypertension (PAH) and renal thrombotic microangiopathy (rTMA) are rare diseases in childhood, frequently leading to death and end-stage renal disease, respectively. Their combined occurrence has been reported anecdotally. We investigated the clinical, biochemical, and genetic aspects of 5 children with the rare combination of PAH and rTMA. Onset of disease ranged from 1.5 to 14 years of age. The 2 youngest patients presented with concomitant pulmonary and renal disease; in the older patients, PAH was preceded by rTMA from age 2.5 to 7 years. Three patients presenting at ≤ 3 years of age died of right ventricular failure secondary to progressive PAH. In 2 patients, cobalamin C (cblC) deficiency was diagnosed postmortem. Three patients were treated with hydroxocobalamin; 1 died 2 weeks after diagnosis, 1 patient exhibited progressive pulmonary vasculopathy, and 1 patient is currently in stable condition. cblC deficiency was diagnosed biochemically 2 days to 18 years after initial presentation. Genetic analysis confirmed mutations in MMACHC in all patients; 4 patients were compound heterozygous, with all having base-pair substitutions (G>A or G>T) at nucleotide 276 in addition to frame-shift mutations. One patient had homozygous nonsense mutations of MMACHC. We established cblC deficiency as the denominator in the rare combination of PAH and rTMA in these children. Early recognition of cblC deficiency and vigorous treatment with hydroxocobalamin may beneficially affect the course of this devastating disease.

The challenge of managing hemophilia A and STEC-induced hemolytic uremic syndrome.

BACKGROUND: The hemolytic uremic syndrome (HUS) is a thrombotic microangiopathy leading to acute kidney injury in children. In most cases it is triggered by an infection caused by Shiga-like toxin-producing Escherichia coli (STEC). Endothelial damage plays a central role in the pathogenesis of disease. Hemophilia A is a genetic disorder leading to factor VIII (FVIII) deficiency, an important factor in the coagulation system. CASE: Here we describe a hemophilia A patient who developed HUS due to a STEC O26 infection. The patient developed not only acute kidney injury, but also severe gastro-intestinal and neurological complications. Increased amounts of recombinant FVIII (rFVIII) had to be administered during the acute phase of the disease to reach acceptable blood levels of FVIII, in order to control the hemorrhagic colitis and to prevent severe neurological complications. CONCLUSION: The patient's treatment schedule of rFVIII during the HUS period was a serious challenge, and we cannot exclude that it contributed to the severity of the HUS by enhancing the thrombotic microangiopathic process. The role of factor VIII administration in the severe outcome of this disease is discussed.