Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes.

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

Spinocerebellar Ataxia Tethering PCR: A Rapid Genetic Test for the Diagnosis of Spinocerebellar Ataxia Types 1, 2, 3, 6, and 7 by PCR and Capillary Electrophoresis.

Spinocerebellar ataxia (SCA) types 1, 2, 3, 6, and 7, associated with a (CAG)n repeat expansion in coding sequences, are the most prevalent autosomal dominant ataxias worldwide (approximately 60% of the cases). In addition, the phenotype of SCA2 expansions has been now extended to Parkinson disease and amyotrophic lateral sclerosis. Their diagnosis is currently based on a PCR to identify small expanded alleles, followed by a second-level test whenever a false normal homozygous or a CAT interruption in SCA1 needs to be verified. Next-generation sequencing still does not allow efficient detection of these repeats. Here, we show the efficacy of a novel, rapid, and cost-effective method to identify and size pathogenic expansions in SCA1, 2, 3, 6, and 7 and recognize large alleles or interruptions without a second-level test. Twenty-five healthy controls and 33 expansion carriers were analyzed: alleles migrated consistently in different PCRs and capillary runs, and homozygous individuals were always distinguishable from heterozygous carriers of both common and large (>100 repeats) pathogenic CAG expansions. Repeat number could be calculated counting the number of peaks, except for the largest SCA2 and SCA7 alleles. Interruptions in SCA1 were always visible. Overall, our method allows a simpler, cost-effective, and sensibly faster SCA diagnostic protocol compared with the standard technique and to the still unadapted next-generation sequencing.

The p.Phe174Ser mutation is associated with mild forms of Smith Lemli Opitz Syndrome.

BACKGROUND: Smith Lemli Opitz syndrome (SLOS; OMIM #270400) is an autosomal recessive metabolic disorder caused by mutations in the DHCR7 gene. SLOS is characterized by a plethora of abnormalities involving mainly the brain and the genitalia but also the cardiac, skeletal and gastroenteric system, typical dysmorphic facial features, and variable degrees of developmental delay and intellectual disability (ID). SLOS has a broad phenotypic spectrum, ranging from multiple congenital malformation syndrome, to mild developmental delay and minor malformations. A large number of mutations have been described in the DHCR7 gene, with few common mutations accounting for the majority of mutated alleles found in patients and a large number of very rare or even private variants. Due to the wide variety of clinical presentations, diagnosis can be difficult, especially in the milder forms of the disorder. Furthermore, establishing a molecular diagnosis can be complicated by finding variants of unknown clinical significance in such cases. CASE PRESENTATION: We report a case of SLOS at the mild end of the clinical spectrum, presenting with bilateral pelvis ectasia, mild dysmorphic features and mild intellectual disability. The case is compound heterozygous for a known pathogenic mutation (c.724C > T, p.Arg242Cys) and a mutation that has only been reported once in a Portuguese patient (c.521 T > C, p.Phe174Ser) whose pathogenicity has not been yet assessed. We compared the two patients carrying the p.Phe174Ser variant and concluded that this variant is associated with mild forms of SLOS. CONCLUSION: We report a patient with a mild case of SLOS, highlighting the importance of recognizing subtle anomalies of the genitourinary system, associated with mild dysmorphic features and mild intellectual disability in establishing the diagnosis of mild forms of SLOS. With this report, we confirm the pathogenicity of the p.Phe174Ser variant and we also provide evidence of its association with mild forms of SLOS. This finding further facilitates the establishment of a genotype-phenotype correlation for SLOS. This helps in counselling for this disorder and in predicting therapeutic responses.

Fcgamma receptor IIA genotype and susceptibility to P. aeruginosa infection in patients with cystic fibrosis.

It has been suggested that genes other than CFTR could modulate the severity of lung disease in cystic fibrosis (CF). Neutrophil Fcgamma receptor II (FcgammaRII) is involved in host defense against microorganisms and in inflammatory response. We evaluated the association between genetic variability of this gene and both airway infection with Pseudomonas aeruginosa and severity of lung disease in patients with CF. We studied 167 Italian unrelated patients with CF and 50 control subjects. The distribution of FcgammaRIIA genotypes in CF patients was compared with that in control subjects and the different genotypes were related with the presence or absence of P. aeruginosa infection and markers of disease severity in CF patients. The distribution of FcgammaRIIA genotypes was not significantly different between CF patients and controls. We observed that in CF patients with the same CFTR genotype (DeltaF508/DeltaF508), those carrying the R allele of FcgammaRIIA had an increased risk of acquiring chronic P. aeruginosa infection (P=0.042, R.R.: 4.38; 95% CI: 1.17/22.4). Moreover, the frequency of R/R genotype in patients with chronic P. aeruginosa infection seems to be higher than that of control subjects and patients without chronic infection. The observation that CF patients carrying the R allele of FcgammaRIIA are at higher risk of acquiring chronic P. aeruginosa infection suggests that the FcgammaRII loci genetic variation is contributing to this infection susceptibility.