BACKGROUND:Short-read genome sequencing (GS) is among the most comprehensive genetic testing methods available, capable of detecting single-nucleotide variants, copy-number variants, mitochondrial variants, repeat expansions, and structural variants in a single assay. Despite its technical advantages, the full clinical utility of GS in real-world diagnostic settings remains to be fully established. METHODS:This study systematically compared singleton GS (sGS), trio GS (tGS), and exome sequencing-based standard-of-care (SoC) genetic testing in 416 patients with rare diseases in a blinded, prospective study. Three independent teams with divergent baseline expertise evaluated the diagnostic yield of GS as a unifying first-tier test and directly compared its variant detection capabilities, learning curve, and clinical feasibility. The SoC team had extensive prior experience in exome-based diagnostics, while the sGS and tGS teams were newly trained in GS interpretation. Diagnostic yield was assessed through both prospective and retrospective analyses. RESULTS:In our prospective analysis, tGS achieved the highest diagnostic yield for likely pathogenic/pathogenic variants at 36.1% in the newly trained team, surpassing the experienced SoC team at 35.1% and the newly trained sGS team at 28.8%. To evaluate which variants could technically be identified and account for differences in team experience, we conducted a retrospective analysis, achieving diagnostic yields of 36.7% for SoC, 39.1% for sGS, and 40.0% for tGS. The superior yield of GS was attributed to its ability to detect deep intronic, non-coding, and small copy-number variants missed by SoC. Notably, tGS identified three de novo variants classified as likely pathogenic based on recent GeneMatcher collaborations and newly published gene-disease association studies. CONCLUSIONS:Our findings demonstrate that GS, particularly tGS, outperforms SoC in diagnosing rare diseases, with sGS providing a more cost-effective alternative. These results suggest that GS should be considered a first-tier genetic test, offering an efficient, single-step approach to reduce the diagnostic odyssey for patients with rare diseases. The trio approach proved especially valuable for less experienced teams, as inheritance data facilitated variant interpretation and maintained high diagnostic yield, while experienced teams achieved comparable results with singleton analysis alone.

AbstractPurposeShort-read genome sequencing (GS) is a comprehensive genetic testing method capable of detecting multiple variant types. Despite its technical advantages, systemic comparisons of singleton GS (sGS), trio GS (tGS), and exome sequencing-based standard-of-care (SoC) in real-world diagnostics remain limited.MethodsWe systematically compared sGS, tGS, and SoC genetic testing in 448 patients with rare diseases in a blinded, prospective study. Three independent teams evaluated the diagnostic yield, variant detection capabilities, and clinical feasibility of GS as a first-tier test. Diagnostic yield was assessed through both prospective and retrospective analyses.ResultsIn prospective analyses, tGS achieved the highest diagnostic yield for likely pathogenic/pathogenic variants (36.8%) in a newly trained team, outperforming the experienced SoC team (36.0%) and the sGS team (30.4%). Retrospective analyses, accounting for technical variant detection and team experience differences, reported diagnostic yields of 38.6% for SoC, 41.3% for sGS, and 42.2% for tGS. GS excelled in identifying deep intronic, non-coding, and small copy-number variants missed by SoC. Notably, tGS additionally identified three de novo variants classified as likely pathogenic based on recent GeneMatcher collaborations and newly published gene-disease association studies.ConclusionGS, particularly tGS, demonstrated superior diagnostic performance, supporting its use as a first-tier genetic test. sGS offers a cost-effective alternative, enabling faster, more efficient diagnoses for rare disease patients.

Recently, an exonic GGC repeat expansion (RE) was identified by long-read genome sequencing in the ZFHX3 gen, causing spinocerebellar ataxia type 4 (SCA4), a dominant form of ataxia with sensory neuropathy. However, the analysis of larger cohorts of patients remained demanding, resulting in a challenge to diagnose patients and leaving the question of anticipation in SCA4 unanswered. We aimed to develop a GGC repeat test for clinical SCA4 screening and to apply this test to screen two large German SCA pedigrees and samples of unrelated patients collected over the last 25 years. We modulated a commercial GGC-RE kit (Bio-Techne AmplideX® Asuragen® PCR/CE FMR1 Reagents) with ZFHX3-specific primers and adapted PCR conditions. The test was applied to patients and 50 healthy controls to determine the exact repeat number. Clinical data were revised and correlated with the expanded allele sizes and an exploratory analysis of structural MRI was performed. Repeat size, determined by our protocol for (GGC)n RE analysis shows a strong inverse correlation between repeat length and age at onset and anticipation in subsequent generations. The phenotype also appears to be more strongly expressed in carriers of longer RE. Clinical red flags were slowed saccades, sensory neuropathy and autonomic dysfunction. Our protocol enables cost-effective and robust screening for the causative SCA4 RE within ZFHX3. Furthermore, detailed clinical data of our patients gives a more precise view on SCA4, which seems to be more common among patients with ataxia than expected.

Mutations affecting the transcriptional regulator Ankyrin Repeat Domain 11 (ANKRD11) are mainly associated with the multisystem developmental disorder known as KBG syndrome, but have also been identified in individuals with Cornelia de Lange syndrome (CdLS) and other developmental disorders caused by variants affecting different chromatin regulators. The extensive functional overlap of these proteins results in shared phenotypical features, which complicate the assessment of the clinical diagnosis. Additionally, re-evaluation of individuals at a later age occasionally reveals that the initial phenotype has evolved toward clinical features more reminiscent of a developmental disorder different from the one that was initially diagnosed. For this reason, variants in ANKRD11 can be ascribed to a broader class of disorders that fall within the category of the so-called chromatinopathies. In this work, we report on the clinical characterization of 23 individuals with variants in ANKRD11. The subjects present primarily with developmental delay, intellectual disability and dysmorphic features, and all but two received an initial clinical diagnosis of either KBG syndrome or CdLS. The number and the severity of the clinical signs are overlapping but variable and result in a broad spectrum of phenotypes, which could be partially accounted for by the presence of additional molecular diagnoses and distinct pathogenic mechanisms.

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B'. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

Correction to: European Journal of Human Genetics doi: 10.1038/ejhg.2011.175; published online 21 September 2011 Since the publication of this article, the authors noted that NIPBL was mistyped in several places. This has now been rectified and the corrected article appears in this issue. The HTML and online PDF versions have also been rectified and now carry the correct name.

The spinocerebellar ataxias (SCAs) with autosomal dominant inheritance are a clinically and genetically heterogeneous group of neurodegenerative disorders. To date 27 different loci have been identified for these conditions. Recently, two deletions as well as one missense mutation in the beta-III spectrin gene (STBN2) were identified causing SCA5. To evaluate the clinical relevance of these mutations, we screened 310 familial and sporadic patients with ataxia. While none of the individuals tested had evidence for one of the known SCA5 mutations, additional sequencing of the coding region for 22 unrelated patients revealed three novel missense exchanges at evolutionary conserved amino acid positions. Even though each variation marks a unique genotype in 250 alleles, a disease causing capacity can be excluded with high probability. These results reflect the challenges for molecular analyses in SCA5.

Background Spinocerebellar ataxia type 17 (SCA17), a neurodegenerative disorder in man, is caused by an expanded polymorphic polyglutamine-encoding trinucleotide repeat in the gene for TATA-box binding protein (TBP), a main transcription factor. Observed pathogenic expansions ranged from 43 – 63 glutamine (Gln) codons (Gln 43–63 ). Reduced penetrance is known for Gln 43–48 alleles. In the vast majority of families with SCA17 an expanded CAG repeat interrupted by a CAA CAG CAA element is inherited stably. Results Here, we report the first pedigree with a Gln 49 allele that is a) not interrupted, b) unstable upon transmission, and c) associated with reduced penetrance or very late age of onset. The 76-year-old father of two SCA17 patients carries the Gln 49 TBP allele but presents without obvious neurological symptoms. His children with Gln 53 and Gln 52 developed ataxia at the age of 41 and 50. Haplotype analysis of this and a second family both with uninterrupted expanded and unstable pathological SCA17 alleles revealed a common core genotype not present in the interrupted expansion of an unrelated SCA17 patient. Review of the literature did not present instability in SCA17 families with expanded alleles interrupted by the CAA CAG CAA element. Conclusion The presence of a Gln 49 SCA17 allele in an asymptomatic 76-year-old male reams the discussion of reduced penetrance and genotypes producing very late disease onset. In SCA17, uninterrupted expanded alleles of TBP are associated with repeat instability and a common founder haplotype. This suggests for uninterrupted expanded alleles a mutation mechanism and some clinical genetic features distinct from those alleles interrupted by a CAA CAG CAA element.

Friedreich's ataxia (FRDA), the most common autosomal recessively inherited ataxia, is due to a homozygous GAA triplet repeat expansion in the first intron of the FRDA gene in about 96% of patients. Approximately 4% of FRDA patients are compound heterozygotes with a GAA repeat expansion in one allele and a point mutation in the coding region of the second allele. To reinvestigate the mutation spectrum, we searched for mutations including exon deletions in six patients heterozygous for the GAA repeat expansion and found two unknown missense mutations, p.Asn146Lys and p.Leu186Arg, in trans to the expanded FRDA allele. Interestingly, we detected a heterozygous 2776 bp deletion including exon 5a in one of our patients. This deletion removes 50 of the 210 residues of the frataxin. Furthermore, since no FRDA case with two-point mutations is known, we screened eight patients with FRDA phenotype but GAA alleles within the normal range but did not reveal a mutation within the FRDA gene. In addition, DNA polymorphisms have been found in four out of 100 control individuals in this study.

The spinocerebellar ataxias (SCAs) with autosomal dominant inheritance are a group of neurodegenerative disorders with overlapping as well as highly variable phenotypes. Genetically, at least 25 different loci have been identified. Seven SCAs are caused by CAG trinucleotide repeat expansions, for 13 the chromosomal localization is known solely. Recently, a missense mutation in the fibroblast growth factor 14 gene (FGF14) has been reported in a Dutch family with a new dominantly inherited form of SCA. To evaluate the frequency of mutations in the FGF14 gene, we performed molecular genetic analyses for the five exons in 208 nonrelated familial ataxia cases and 208 control samples. In one patient, we detected a novel single base pair deletion in exon 4 (c.487delA) creating a frameshift mutation. In addition, we found DNA polymorphisms in exon 1a, 4, and 5, an amino-acid exchange at position 124, as well as a single-nucleotide polymorphism in the 3′-untranslated region of exon 5.

Ciliary neurotrophic factor (CNTF), originally known for its involvement in the modulation of neuronal growth, has been discovered to exert anorexigenic effects and is currently being investigated in clinical studies for the treatment of obesity and insulin resistance. This neuropeptide acts on the central nervous system. However, we have recently demonstrated direct peripheral effects on adipocyte signalling and thermogenesis. Given the emerging endocrine role of adipose tissue in the regulation of energy homeostasis and insulin resistance, we investigated potential effects of CNTF on leptin expression and secretion. Our study demonstrates a direct inhibition of leptin expression and secretion by acute and chronic CNTF treatment. Furthermore, we demonstrate a differentiation- and Janus kinase 2 (JAK2)-independent, but phosphatidylinositol 3-kinase-dependent signalling pathway mediating this negative effect. These results provide novel evidence for a role of CNTF in the selective modulation of adipocyte endocrine function which may have important implications for the regulation of energy homeostasis.