Reverse Phenotyping after Whole-Exome Sequencing in Children with Developmental Delay/Intellectual Disability-An Exception or a Necessity?

This study delves into the diagnostic yield of whole-exome sequencing (WES) in pediatric patients presenting with developmental delay/intellectual disability (DD/ID), while also exploring the utility of Reverse Phenotyping (RP) in refining diagnoses. A cohort of 100 pediatric patients underwent WES, yielding a diagnosis in 66% of cases. Notably, RP played a significant role in cases with negative prior genetic testing, underscoring its significance in complex diagnostic scenarios. The study revealed a spectrum of genetic conditions contributing to DD/ID, illustrating the heterogeneity of etiological factors. Despite challenges, WES demonstrated effectiveness, particularly in cases with metabolic abnormalities. Reverse phenotyping was indicated in half of the patients with positive WES findings. Neural network models exhibited moderate-to-exceptional predictive abilities for aiding in patient selection for WES and RP. These findings emphasize the importance of employing comprehensive genetic approaches and RP in unraveling the genetic underpinnings of DD/ID, thereby facilitating personalized management and genetic counseling for affected individuals and families. This research contributes insights into the genetic landscape of DD/ID, enhancing our understanding and guiding clinical practice in this particular field of clinical genetics.

Premutations in the FMR1 gene in Serbian patients with undetermined tremor, ataxia and parkinsonism.

Introduction: Although one of the most common monogenic late-onset neurodegenerative disorders, fragile-X-associated tremor/ataxia syndrome (FXTAS) is still underdiagnosed. The aim of the present study was to estimate the frequency of premutation carriers in patients with unexplained degenerative ataxias, action tremor or parkinsonism, and action tremor with or without associated cognitive impairment.Methods: The study comprised 100 consecutive patients with the disease onset >49 years who had any form of unexplained action tremor, cerebellar ataxia, followed by parkinsonism with or without incipient dementia, and in whom the FMR1 repeats size was determined.Results: Premutation in the FMR1 was identified in two patients (2%): the first, male patient had 83 CGG repeats and the second, female patient had 32 and 58 CGG repeats.Discussion/Conclusion: FXTAS was relatively rare among older patients with unexplained ataxia and action tremor, with or without parkinsonism and/or cognitive impairment. Tremor and ataxia were major clinical features in our two patients, although parkinsonism, autonomic dysfunction and psychiatric problems might be an important part of the spectrum. Probable FXTAS should be considered in the differential diagnosis of patients with unexplained action tremor and ataxia, and undetermined parkinsonism, especially when there was a positive family history for involuntary movement disorders in other family members and/or autism spectrum disorders in younger cousins.

Novel intragenic deletions within the UBE3A gene in two unrelated patients with Angelman syndrome: case report and review of the literature.

BACKGROUND: Patients with Angelman syndrome (AS) are affected by severe intellectual disability with absence of speech, distinctive dysmorphic craniofacial features, ataxia and a characteristic behavioral phenotype. AS is caused by the lack of expression in neurons of the UBE3A gene, which is located in the 15q11.2-q13 imprinted region. Functional loss of UBE3A is due to 15q11.2-q13 deletion, mutations in the UBE3A gene, paternal uniparental disomy and genomic imprinting defects. CASE PRESENTATION: We report here two patients with clinical features of AS referred to our hospital for clinical follow-up and genetic diagnosis. Methylation Specific-Multiplex Ligation-Dependent Probe Amplification (MS-MLPA) of the 15q11.2-q13 region was carried out in our laboratory as the first diagnostic tool detecting two novel UBE3A intragenic deletions. Subsequently, the MLPA P336-A2 kit was used to confirm and determine the size of the UBE3A deletion in the two patients. A review of the clinical features of previously reported patients with whole UBE3A gene or partial intragenic deletions is presented here together with these two new patients. CONCLUSION: Although rare, UBE3A intragenic deletions may represent a small fraction of AS patients without a genetic diagnosis. Testing for UBE3A intragenic exonic deletions should be performed in those AS patients with a normal methylation pattern and no mutations in the UBE3A gene.

Chromosomal instability in patients with Fanconi anemia from Serbia.

BACKGROUND/AIM: Fanconi anemia (FA) is a rare hereditary disease in a heterogeneous group of syndromes, so-called chromosome breakage disorders. Specific hypersensitivity of its cells to chemical agents, such as diepoxybutane (DEB), was used as a part of screening among patients with clinical suspicion of FA. The aim of this study was to determine chromosomal instability in patients with FA symptoms in Serbia. METHODS: A total of 70 patients with phenotypic symptoms of FA, diagnosed at the Mother and Child Health Care Institute of Serbia "Dr Vukan Cupic, Belgrade and University Children's Hospital, Belgrade from February 2004 to September 2011, were included in this study. Cytogenetic instability analysis was performed on untreated and DEB-treated 72 h-cultures of peripheral blood. RESULTS: Ten patients in the group of 70 suspected of FA, showed increased DEB induced chromosome breakage and were classified into the FA group. The range of DEB induced aberrant cells percentages in the FA group was from 32% to 82%. DEB sensitivity of 58 tested patients were bellow FA values (range: 0-6%) (non-FA group), with no overlapping. The remaining two patients showed borderline sensitivity (borderline FA group - FA*), comparing to the healthy controls. CONCLUSION: This study revealed 10 patients with FA on the basis of cytogenetic analysis of DEB induced chromosome aberrations. Our results are in consistency with those from the literature. Early and precise diagnosis of FA is very important in further treatment of these patients, considering its cancer prone and lethal effects.

Ten years of experience in molecular prenatal diagnosis and carrier testing for spinal muscular atrophy among families from Serbia.

OBJECTIVE: To describe 10 years of experience of prenatal analysis of spinal muscular atrophy (SMA). METHODS: Data were retrospectively evaluated from prenatal analysis and carrier screening among parents and close relatives between January 2003 and December 2012. Screening was done before the parents were offered prenatal diagnosis. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to detect the most frequent homozygous deletions in the SMN1 gene in fetal samples. A commercial MLPA kit (SALSA P060) was used to analyze SMN1 copy number for carrier status determination among healthy individuals. Bayesian calculation was used to accurately assess the risk of having a child affected with SMA. RESULTS: During the study period, 66 fetal samples from 44 Serbian families were analyzed, and 13 (19.7%) showed a homozygous deletion in the SMN1 gene. Among 28 healthy individuals, carrier status was confirmed for 16 (57.1%). For 7 couples, quantitative analyses and Bayesian calculation reduced the final risk of having a child with SMA from 1 in 200 to 1 in 2448. CONCLUSION: Owing to disease severity and lack of a curative treatment, prenatal diagnosis of SMA is the best way to prevent recurrence. Carrier detection allows accurate risk assessment and appropriate genetic counseling for all family members.

Complex small supernumerary marker chromosomes - an update.

BACKGROUND: Complex small supernumerary marker chromosomes (sSMC) constitute one of the smallest subgroups of sSMC in general. Complex sSMC consist of chromosomal material derived from more than one chromosome; the best known representative of this group is the derivative chromosome 22 {der(22)t(11;22)} or Emanuel syndrome. In 2008 we speculated that complex sSMC could be part of an underestimated entity. RESULTS: Here, the overall yet reported 412 complex sSMC are summarized. They constitute 8.4% of all yet in detail characterized sSMC cases. The majority of the complex sSMC is contributed by patients suffering from Emanuel syndrome (82%). Besides there are a der(22)t(8;22)(q24.1;q11.1) and a der(13)t(13;18)(q11;p11.21) or der(21)t(18;21)(p11.21;q11.1) = der(13 or 21)t(13 or 21;18) syndrome. The latter two represent another 2.6% and 2.2% of the complex sSMC-cases, respectively. The large majority of complex sSMC has a centric minute shape and derives from an acrocentric chromosome. Nonetheless, complex sSMC can involve material from each chromosomal origin. Most complex sSMC are inherited form a balanced translocation in one parent and are non-mosaic. Interestingly, there are hot spots for the chromosomal breakpoints involved. CONCLUSIONS: Complex sSMC need to be considered in diagnostics, especially in non-mosaic, centric minute shaped sSMC. As yet three complex-sSMC-associated syndromes are identified. As recurrent breakpoints in the complex sSMC were characterized, it is to be expected that more syndromes are identified in this subgroup of sSMC. Overall, complex sSMC emphasize once more the importance of detailed cytogenetic analyses, especially in patients with idiopathic mental retardation.

Lower incidence of deletions in the survival of motor neuron gene and the neuronal apoptosis inhibitory protein gene in children with spinal muscular atrophy from Serbia.

Spinal muscular atrophy (SMA) is the second most frequent autosomal recessive disease characterized by degeneration of the anterior horn cells of the spinal cord, leading to muscular atrophy. SMA is classified into three types according to disease severity and age-onset: severe (type I), intermediate (type II) and mild (type III). Deletions in the survival motor neuron (SMN) gene, located in the chromosome region 5q11.2- 5q13.3, are major determinants of SMA phenotype. Extended deletions that include the neuronal apoptosis inhibitory protein (NAIP) gene may correlate with the severtity of SMA. SMN gene is present in two highly homologous copies, SMN1 and SMN2, but only deletions of the SMN1 gene (exons 7 and 8 or exon 7) are responsible for clinical manifestations of SMA. Here, we present the deletion profiling of SMN1 and NAIP genes in 89 children with SMA from Serbia: 52 patients with type I, 26 with type II, and 11 with type III. The homozygous deletion of the SMN1 gene was confirmed in 72 of 89 (81%) patients, being the most frequent in SMA type I (48/52): 68 patients (94.4%) with deletion of exons 7 and 8 and 4 patients (5.6%) with deletion of exon 7. The extended deletion including the NAIP gene was detected in 18 of 89 (20.2%) patients, mostly affected with type I. This study has revealed the lower incidence of deletions in the SMN1 and NAIP genes in families with SMA in Serbia and will provide important information for genetic counselling in these families.

Successful treatment of hodgkin lymphoma in nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS) is a rare DNA repair disorder characterized by microcephaly, immunodeficiency, and predisposition to malignancy. We report on a 5-year-old patient with NBS who presented with nodular sclerosing type of Hodgkin disease stage IVB. Chemotherapy consisting of COPP/ABV regimen with reduction at 75% of full doses was employed. During this treatment, no major toxic or infectious complications were observed. Complete remission was achieved lasting now for 20 months. In DNA repair disorders, prognosis of Hodgkin disease is poor as opposed to excellent overall prognosis in general pediatric population. Better survival may be achieved both with adopted, disease-specific regimens, and individualized approach considering patient's clinical condition. Also, better recognition and treatment of infections during chemotherapy may reduce early deaths in patients with DNA repair disorders.