Improved molecular diagnosis of patients with neonatal diabetes using a combined next-generation sequencing and MS-MLPA approach.

BACKGROUND: We evaluated a methylation-specific multiplex-ligation-dependent probe amplification (MS-MLPA) assay for the molecular diagnosis of transient neonatal diabetes mellitus (TNDM) caused by 6q24 abnormalities and assessed the clinical utility of using this assay in combination with next generation sequencing (NGS) analysis for diagnosing patients with neonatal diabetes (NDM). METHODS: We performed MS-MLPA in 18 control samples and 42 retrospective NDM cases with normal bi-parental inheritance of chromosome 6. Next, we evaluated 22 prospective patients by combining NGS analysis of 11 NDM genes and the MS-MLPA assay. RESULTS: 6q24 aberrations were identified in all controls and in 19% of patients with normal bi-parental inheritance of chromosome 6. The MS-MLPA/NGS combined approach identified a genetic cause in ~64% of patients with NDM of unknown etiology. CONCLUSIONS: MS-MLPA is a reliable method to identify all known 6q24 abnormalities and comprehensive testing of all causes reveals a causal mutation in ~64% of patients.

Two novel RAD21 mutations in patients with mild Cornelia de Lange syndrome-like presentation and report of the first familial case.

Cornelia de Lange syndrome (CdLS) is a developmental disorder characterized by limb reduction defects, characteristic facial features and impaired cognitive development. Mutations in the NIPBL gene predominate; however, mutations in other cohesin complex genes have also been implicated, particularly in atypical and mild CdLS cases. Missense mutations and whole gene deletions in RAD21 have been identified in children with growth retardation, minor skeletal anomalies and facial features that overlap findings in individuals with CdLS. We report the first intragenic deletion and frameshift mutations identified in RAD21 in two patients presenting with atypical CdLS. One patient had an in-frame deletion of exon 13, while the second patient had a c.592_593dup frameshift mutation. The first patient presented with developmental delay, hypospadias, inguinal hernia and dysmorphic features while, the second patient presented with developmental delay, characteristic facial features, hirsutism, and hand and feet anomalies, with the first patient being milder than the second. The in-frame deletion mutation was found to be inherited from the mother who had a history of melanoma and other unspecified medical problems. This study expands the spectrum of RAD21 mutations and emphasizes the clinical utility of performing RAD21 mutation analysis in patients presenting with atypical forms of CdLS. Moreover, the variability of clinical presentation within families and low penetrance of mutations as well as the significance of performing molecular genetic testing in mildly affected patients are discussed.

MCPH1 deletion in a newborn with severe microcephaly and premature chromosome condensation.

A newborn with severe microcephaly and a history of parental consanguinity was referred for cytogenetic analysis and subsequently for genetic evaluation. While a 46,XY karyotype was eventually obtained, premature chromosome condensation was observed. A head MRI confirmed primary microcephaly. This combination of features focused clinical interest on the MCPH1 gene and directed genetic testing by sequence analysis and duplication/deletion studies disclosed a homozygous deletion of exons 1-11 of the MCPH1 gene. This case illustrates a strength of standard cytogenetic evaluation in directing molecular testing to a single target gene in this disorder, allowing much more rapid diagnosis at a substantial cost savings for this family.