PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24% P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

The standard of care for first-tier clinical investigation of the aetiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy-number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion–deletions (indels) and single-nucleotide variant (SNV) mutations. Whole-genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilised WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a fourfold increase in diagnostic rate over CMA (8%; P value=1.42E−05) alone and more than twofold increase in CMA plus targeted gene sequencing (13%; P value=0.0009). WGS identified all rare clinically significant CNVs that were detected by CMA. In 26 patients, WGS revealed indel and missense mutations presenting in a dominant (63%) or a recessive (37%) manner. We found four subjects with mutations in at least two genes associated with distinct genetic disorders, including two cases harbouring a pathogenic CNV and SNV. When considering medically actionable secondary findings in addition to primary WGS findings, 38% of patients would benefit from genetic counselling. Clinical implementation of WGS as a primary test will provide a higher diagnostic yield than conventional genetic testing and potentially reduce the time required to reach a genetic diagnosis.

The screening by MS/MS for up to 23 additional disorders has approximately doubled the detection rate compared with that achieved by the conventional methods used in Germany. This strategy represents valuable preventive medicine by enabling diagnosis and treatment before the onset of symptoms.

Contrary to many other viruses, the initial steps of the hepatitis B virus (HBV) infection, including attachment to hepatocytes, specific receptor interactions, and membrane fusion, are unsolved. Using HepaRG cells as an in vitro cell culture system, we here report that HBV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cell-surface-associated heparan sulfate proteoglycans. Binding to GAGs requires the integrity of the pre-S domain as a part of the large (L-) viral envelope protein. H epatitis B virus (HBV) represents the medically important prototype of a family of small, enveloped DNA viruses (hepadnaviridae), which are widespread in mammals and birds. All members of this family possess high species and, at least with respect to the preferential site of replication, also tissue specificity. 1 Hepadnaviral infections cause transient or persistent liver inflammation, with approximately 360 million people worldwide being chronic HBV carriers and approximately 650,000 deaths each year attributable to HBVrelated progressive liver failure (cirrhosis or hepatocellular carcinoma). 2 For a long time, in vitro studies of the HBV life cycle, particularly the early infection events (attachment, receptor binding, and fusion), were only feasible using primary human hepatocytes (PHH). The application of PHH was, however, limited through restrictions in accessibility and high variations in susceptibility to HBV infection. 3 The establishment of the highly differentiable human hepatoma cell line HepaRG resolved this issue and facilitated the systematic analysis of the early HBV infection events. 4

We report studies of a Croatian boy, a proven case of human S-adenosylhomocysteine (AdoHcy) hydrolase deficiency. Psychomotor development was slow until his fifth month; thereafter, virtually absent until treatment was started. He had marked hypotonia with elevated serum creatine kinase and transaminases, prolonged prothrombin time and low albumin. Electron microscopy of muscle showed numerous abnormal myelin figures; liver biopsy showed mild hepatitis with sparse rough endoplasmic reticulum. Brain MRI at 12.7 months revealed white matter atrophy and abnormally slow myelination. Hypermethioninemia was present in the initial metabolic study at age 8 months, and persisted (up to 784 M) without tyrosine elevation. Plasma total homocysteine was very slightly elevated for an infant to 14.5-15.9 M. In plasma, S-adenosylmethionine was 30-fold and AdoHcy 150-fold elevated. Activity of AdoHcy hydrolase was Ϸ3% of control in liver and was 5-10% of the control values in red blood cells and cultured fibroblasts. We found no evidence of a soluble inhibitor of the enzyme in extracts of the patient's cultured fibroblasts. Additional pretreatment abnormalities in plasma included low concentrations of phosphatidylcholine and choline, with elevations of guanidinoacetate, betaine, dimethylglycine, and cystathionine. Leukocyte DNA was hypermethylated. Gene analysis revealed two mutations in exon 4: a maternally derived stop codon, and a paternally derived missense mutation. We discuss reasons for biochemical abnormalities and pathophysiological aspects of AdoHcy hydrolase deficiency.

Pneumonia and systemic infection are common in premature infants. The antimicrobial peptides human beta-defensin 1 and 2 (hBD-1 and hBD-2) and the cathelicidin LL-37/hCAP-18 are effector molecules of the innate respiratory immune system. It is unknown whether these host defense substances are produced in the respiratory tract of newborns. Concentrations of these peptides were determined in tracheal aspirates of mechanically ventilated newborn infants. All three antimicrobial peptides could be detected in airway lining fluid with equivalent levels in term and preterm newborns. Concentrations of antimicrobial peptides correlated with each other and with levels of interleukin-8 and tumor necrosis factor-alpha in the bronchoalveolar lavage fluid. Pulmonary or systemic infections were associated with significantly increased concentrations of LL-37, hBD-1, and hBD-2. Western blotting detected mature peptides in the lavage fluid. In conclusion, mucosal antimicrobial peptides are present in lung secretions of premature and mature newborns. The molecules are upregulated in response to infection and inflammation and probably represent effector molecules of the respiratory defense system.

Previous studies showed that the N-terminal 75 amino acids of the pre-S1 domain of the hepatitis B virus (HBV) L protein are essential for HBV and hepatitis delta virus (HDV) infectivity. Consistently, synthetic lipopeptides encompassing this sequence or only parts of it efficiently block HBV and HDV infection, presumably through specific interference with a cellular receptor. Crucial for both virus infectivity and the inhibitory activity of the peptides are N-terminal myristoylation and a highly conserved motif within the N-terminal 48 amino acids. To refine the sequence requirements, we synthesized a series of HBV pre-S1 peptides containing deletions, point mutations, D-amino acid exchanges, or genotype-specific sequence permutations. Using the HepaRG cell line and a genotype D-derived virus, we determined the specific inhibitory activities of the peptides and found that (i) lipopeptides with an artificial consensus sequence inhibit HBV genotype D infection more potently than the corresponding genotype D peptides; (ii) point mutations, D-amino acid exchanges, or deletions introduced into the highly conserved part of the pre-S1 domain result in an almost complete loss of activity; and (iii) the flanking sequences comprising amino acids 2 to 8, 16 to 20, and, to a less pronounced extent, 34 to 48 gradually increase the inhibitory activity, while amino acids 21 to 33 behave indifferently. Taken together, our data suggest that HBV pre-S1-mediated receptor interference and, thus, HBV receptor recognition form a highly specific process. It requires an N-terminal acyl moiety and a highly conserved sequence that is present in primate but not rodent or avian hepadnaviruses, indicating different entry pathways for the different family members.Hepatitis B viruses (HBVs) are small, enveloped DNA viruses that replicate their genome via reverse transcription of a pregenomic RNA transcript in the cytoplasm of infected hepatocytes (31). They are classified into the family Hepadnaviridae and are adapted to mammals (primates and rodents) and birds, where they cause acute and persistent infections. At present about 360 million people are chronically infected with the human HBV. Due to HBV-related progressive liver failure (cirrhosis or hepatocellular carcinoma) Ϸ650,000 people die each year (10). HBV, like duck hepatitis B virus (DHBV) or woodchuck hepatitis B virus (WHV), shows a pronounced species specificity and a remarkable liver tropism. These features have been assigned to specific early infection events, most likely receptor recognition. However, it cannot be excluded that other steps during or postentry contribute to the host restrictions of infection (13).The HBV replication cycle has been deciphered in some detail. However, studies on the early infection events were hampered by the lack of appropriate in vitro infection systems. The establishment of the HBV-susceptible cell line HepaRG and systems based on primary human hepatocytes (PHH) and primary Tupaia belangeri hepatocytes resolved this issue and facilitated inves...

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