Long read sequencing on its way to the routine diagnostics of genetic diseases.

The clinical application of technological progress in the identification of DNA alterations has always led to improvements of diagnostic yields in genetic medicine. At chromosome side, from cytogenetic techniques evaluating number and gross structural defects to genomic microarrays detecting cryptic copy number variants, and at molecular level, from Sanger method studying the nucleotide sequence of single genes to the high-throughput next-generation sequencing (NGS) technologies, resolution and sensitivity progressively increased expanding considerably the range of detectable DNA anomalies and alongside of Mendelian disorders with known genetic causes. However, particular genomic regions (i.e., repetitive and GC-rich sequences) are inefficiently analyzed by standard genetic tests, still relying on laborious, time-consuming and low-sensitive approaches (i.e., southern-blot for repeat expansion or long-PCR for genes with highly homologous pseudogenes), accounting for at least part of the patients with undiagnosed genetic disorders. Third generation sequencing, generating long reads with improved mappability, is more suitable for the detection of structural alterations and defects in hardly accessible genomic regions. Although recently implemented and not yet clinically available, long read sequencing (LRS) technologies have already shown their potential in genetic medicine research that might greatly impact on diagnostic yield and reporting times, through their translation to clinical settings. The main investigated LRS application concerns the identification of structural variants and repeat expansions, probably because techniques for their detection have not evolved as rapidly as those dedicated to single nucleotide variants (SNV) identification: gold standard analyses are karyotyping and microarrays for balanced and unbalanced chromosome rearrangements, respectively, and southern blot and repeat-primed PCR for the amplification and sizing of expanded alleles, impaired by limited resolution and sensitivity that have not been significantly improved by the advent of NGS. Nevertheless, more recently, with the increased accuracy provided by the latest product releases, LRS has been tested also for SNV detection, especially in genes with highly homologous pseudogenes and for haplotype reconstruction to assess the parental origin of alleles with de novo pathogenic variants. We provide a review of relevant recent scientific papers exploring LRS potential in the diagnosis of genetic diseases and its potential future applications in routine genetic testing.

Chromosomal Microarray Analysis Identifies a Novel SALL1 Deletion, Supporting the Association of Haploinsufficiency with a Mild Phenotype of Townes-Brocks Syndrome.

SALL1 heterozygous pathogenic variants cause Townes-Brocks syndrome (TBS), a condition with variable clinical presentation. The main features are a stenotic or imperforate anus, dysplastic ears, and thumb malformations, and other common concerns are hearing impairments, foot malformations, and renal and heart defects. Most of the pathogenic SALL1 variants are nonsense and frameshift, likely escaping nonsense-mediated mRNA decay and causing disease via a dominant-negative mechanism. Haploinsufficiency may result in mild phenotypes, but only four families with distinct SALL1 deletions have been reported to date, with a few more being of larger size and also affecting neighboring genes. We report on a family with autosomal dominant hearing impairment and mild anal and skeletal anomalies, in whom a novel 350 kb SALL1 deletion, spanning exon 1 and the upstream region, was identified by array comparative genomic hybridization. We review the clinical findings of known individuals with SALL1 deletions and point out that the overall phenotype is milder, especially when compared with individuals who carry the recurrent p.Arg276Ter mutation, but with a possible higher risk of developmental delay. Chromosomal microarray analysis is still a valuable tool in the identification of atypical/mild TBS cases, which are likely underestimated.

DNAJB11 Mutation in ADPKD Patients: Clinical Characteristics in a Monocentric Cohort.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a late-onset cilia-related disorder, characterized by progressive cystic enlargement of the kidneys. It is genetically heterogeneous with PKD1 and PKD2 pathogenic variants identified in approximately 78% and 15% of families, respectively. More recently, additional ADPKD genes, such as DNAJB11, have been identified and included in the diagnostic routine test for renal cystic diseases. However, despite recent progress in ADPKD molecular approach, approximately ~7% of ADPKD-affected families remain genetically unresolved. We collected a cohort of 4 families from our center, harboring heterozygous variants in the DNAJB11 gene along with clinical and imaging findings consistent with previously reported features in DNAJB11 mutated patients. Mutations were identified as likely pathogenetic (LP) in three families and as variants of uncertain significance (VUS) in the remaining one. One patient underwent to kidney biopsy and showed a prevalence of interstitial fibrosis that could be observed in ~60% of the sample. The presence in the four families from our cohort of ADPKD characteristics together with ADTKD features, such as hyperuricemia, diabetes, and chronic interstitial fibrosis, supports the definition of DNAJB11 phenotype as an overlap disease between these two entities, as originally suggested by the literature.

One-carbon pathway and cognitive skills in children with Down syndrome.

This work investigates the role of metabolite levels in the intellectual impairment of subjects with Down syndrome (DS). Homocysteine, folate, vitamin B12, uric acid (UA), creatinine levels and MTHFR C677T genotype were analyzed in 147 subjects with DS. For 77 subjects, metabolite levels were correlated with cognitive tests. Griffiths-III test was administered to 28 subjects (3.08-6.16 years) and WPPSI-III test was administered to 49 subjects (7.08-16.08 years). Significant correlations were found among some metabolite levels and between homocysteine levels and MTHFR C677T genotype. Moreover, homocysteine, UA and creatinine levels resulted increased with age. We did not find any correlation between metabolites and cognitive test score in the younger group. Homocysteine showed statistically significant correlation with WPPSI-III subtest scores when its level is ≥ 7.35 µmol/L, remaining correlated in higher thresholds only for non-verbal area scores. Vitamin B12 showed correlations with all WPPSI-III subtest scores when its level is < 442 pg/mL. The relevance of the present findings is the detection of a specific metabolite threshold related with a better or worse cognitive score, suggesting that vitamin B12 and homocysteine may have a role in cognitive development in children with DS.

MTHFR C677T polymorphism analysis: A simple, effective restriction enzyme-based method improving previous protocols.

BACKGROUND: 5,10-Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR-RFLP method for the detection of this polymorphism. METHODS: In order to retrieve all published articles possibly describing any PCR-RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer-BLAST software was used to check primer pair's biological specificity. RESULTS: The analysis of previous literature showed that PCR-RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA-fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by Hinf I enzyme in two pieces of 146 bp and 367 bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out. CONCLUSION: Our original PCR-RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR-RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date.