Margherita Baldassarri

Revealing the Complexity of a Monogenic Disease: Rett Syndrome Exome Sequencing

Rett syndrome (OMIM#312750) is a monogenic disorder that may manifest as a large variety of phenotypes ranging from very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005) mutation and balanced X-inactivation, one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome), while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant). In addition to the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system, exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

Interstitial 22q13 deletions not involving SHANK3 gene: A new contiguous gene syndrome

Phelan–McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan–McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan–McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan–McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region.

Coffin-Siris and Nicolaides-Baraitser syndromes are a common well recognizable cause of intellectual disability.

BACKGROUND Nicolaides-Baraitser and Coffin-Siris syndromes are emerging conditions with overlapping clinical features including intellectual disability and typical somatic characteristics, especially sparse hair, low frontal hairline, large mouth with thick and everted lips, and hands and feet anomalies. Since 2012, mutations in genes encoding six proteins of the BAF complex were identified in both conditions. METHODS AND RESULTS We have clinically evaluated a cohort of 1161 patients with intellectual disability from three different Italian centers. A strong clinical suspicion of either Nicolaides-Baraitser syndrome or Coffin-Siris syndrome was proposed in 11 cases who were then molecularly confirmed: 8 having de novo missense mutations in SMARCA2, two frame-shift mutations in ARID1B and one missense mutation in SMARCB1. Given the high frequency of the condition we set up a one-step deep sequencing test for all 6 genes of the BAF complex. CONCLUSIONS These results prove that the frequency of these conditions may be as high as the most common syndromes with intellectual deficit (about 1%). Clinical geneticists should be well aware of this group of disorders in the clinical setting when ascertaining patients with intellectual deficit, the specific facial features being the major diagnostic handle. Finally, this work adds information on the clinical differences of the two conditions and presents a fast and sensitive test for the molecular diagnosis.

Alport syndrome: impact of digenic inheritance in patients management

Alport syndrome (ATS) is a genetically heterogeneous nephropathy with considerable phenotypic variability and different transmission patterns, including monogenic (X‐linked/autosomal) and digenic inheritance (DI). Here we present a new series of families with DI and we discuss the consequences for genetic counseling and risk assessment. Out of five families harboring variants in more than one COL4 gene detected by next generation sequencing (NGS), minigene‐splicing assay allowed us to identify four as true digenic. Two families showed COL4A3/A4 mutations in cis, mimicking an autosomal dominant inheritance with a more severe phenotype and one showed COL4A3/A4 mutations in trans, mimicking an autosomal recessive inheritance with a less severe phenotype. In a fourth family, a de novo mutation (COL4A5) combined with an inherited mutation (COL4A3) triggered a more severe phenotype. A fifth family, predicted digenic on the basis of silico tools, rather showed monogenic X‐linked inheritance due to a hypomorphic mutation, in accordance with a milder phenotype. In conclusion, this study highlights the impact of DI in ATS and explains the associated atypical presentations. More complex inheritance should be therefore considered when reviewing prognosis and recurrence risks. On the other side, these findings emphasize the importance to accompany NGS with splicing assays in order to avoid erroneous identification of at risk members.

Omic Approach in Non-smoker Female with Lung Squamous Cell Carcinoma Pinpoints to Germline Susceptibility and Personalized Medicine

Purpose Lung cancer is strongly associated to tobacco smoking. However, global statistics estimate that in females the proportion of lung cancer cases that is unrelated to tobacco smoking reaches fifty percent, making questionable the etiology of the disease. Materials and Methods A never-smoker female with primary EGFR/KRAS/ALK-negative squamous cell carcinoma of the lung and their normal sibswere subjected to a novel integrative “omic” approach using a pedigree-based model for discovering genetic factors leading to cancer in the absence of well-known environmental trigger. A first-stepwhole-exome sequencing on tumor and normal tissue did not identify mutations in known driver genes. Building on the idea of a germline oligogenic origin of lung cancer, we performed whole-exome sequencing of DNA from patients’ peripheral blood and their unaffected sibs. Finally, RNA-sequencing analysis in tumoral and matched non-tumoral tissues was carried out in order to investigate the clonal profile and the pathogenic role of the identified variants. Results Filtering for rare variants with Combined Annotation Dependent Depletion (CADD) > 25 and potentially damaging effect, we identified rare/private germline deleterious variants in 11 cancer-associated genes, none ofwhich, except one, sharedwith the healthy sib, pinpointing to a “private” oligogenic germline signature. Noteworthy, among these, two mutated genes, namely ACACA and DEPTOR, turned to be potential targets for therapy because related to known drivers, such as BRCA1 and EGFR. Conclusion In the era of precision medicine, this report emphasizes the importance of an “omic” approach to uncover oligogenic germline signature underlying cancer development and to identify suitable therapeutic targets as well.

Combined ultrasound and exome sequencing approach recognizes Opitz G/BBB syndrome in two malformed fetuses.

Orofacial clefts are the most common congenital craniofacial anomalies and can occur as an isolated defect or be associated with other anomalies such as posterior fossa anomalies as a part of several genetic syndromes. We report two consecutive voluntary pregnancy interruptions in a nonconsanguineous couple following the fetal ultrasound finding of cleft lip and palate and posterior fossa anomalies confirmed by means of post-termination examination on the second fetus. The quantitative fluorescent PCR, the karyotype, and the comparative genomic hybridization-array analysis after amniocentesis were normal. Exome sequencing on abortive material from both fetuses detected a missense mutation in MID1, resulting in a clinical diagnosis of Opitz G/BBB syndrome. The same mutation was found in the mother and in her brother, who both revealed cerebellar anomalies at an MRI examination. Our study supports the efficacy of exome sequencing in the presence of both a family history suggestive of an inherited disorder and well-documented ultrasound findings. It reveals the importance of a synergistic effort between gynecologists and geneticists aimed at the integration of the most sophisticated ultrasound techniques with the next-generation sequencing tools to provide a definite diagnosis essential to orient the final decision and to estimate a proper recurrence risk.

Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome

Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end‐stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes‐targeted therapeutic strategies have been hampered by the difficulty of non‐invasively isolating them and transcripts‐based diagnostic approaches are complicated by the inaccessibility of other COL4 chains‐expressing cells. We firstly isolated podocyte‐lineage cells from ATS patients’ urine samples, in a non‐invasive way. RT‐PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patients urine derived podocyte‐lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease‐relevant cells.

CKAP2L mutation confirms the diagnosis of Filippi syndrome

To the Editor: Procedures employed followed the principles outlined in the Helsinki Declaration. The parents of the child provided written consent to publication of material (including pictures) about the patient in the Journal. Filippi syndrome is a rare craniodigital syndrome with autosomal recessive inheritance. Main clinical findings include microcephaly, syndactyly of fingers and toes, peculiar facial features, intellectual disability with a predominant impairment of speech area and growth delay. We present a child that came to our observation for the first time at the age of 8 months for a clinical evaluation regarding microcephaly, facial dysmorphisms and growth delay. He was the second child of Sardinian unrelated parents. He was born at 40 + 2 weeks from spontaneous delivery after an uneventful pregnancy. Auxological parameter percentiles (ct) at birth were: body weight 5th to 10th ct, length 25th ct and head circumference 3rd ct. Perinatal period was regular but a light delay in reaching developmental milestones was noticed. Clinical evaluation at 8 months revealed the following auxological measurement percentiles: body weight −3 SD, length −2 SD, head circumference −3.5 SD. Physical examination documented: plagiocephaly, thin hair with high hairline, arched eyebrows, long eyelashes, large ear pinnae with irregular left helix, wide nasal bridge, bulbous nasal tip with hypoplastic alae nasi, long phyltrum, thin slightly asymmetric lips, small wide-spaced teeth and pointed chin (Figure 1). He also presented bilateral fifth finger clinodactily, left singular palmar crease, bilateral syndactyly of second, third, fourth toes, dystrophic toes nails and left cryptorchydysm. Investigations for developmental delay were planned. Brain Magnetic Resonance Imaging (MRI) did not document any abnormal finding, Electroencephalography (EEG) recording showed poor electrical organization and metabolic tests were within reference range. Diagnostic workup for short stature was performed: hand radiography (RX) showed a delayed bone age, while arginine challenge test documented a Growth Hormone (GH) deficiency; therefore, GH replacement therapy was started. The accurate analysis of the phenotypic details presented by the patient led us to suspect Filippi syndrome. The first case of this syndrome was described in 1985 by Filippi who identified this new autosomal recessive condition in 3 of 8 Sardinian siblings born from healthy unrelated parents. The affected individuals had striking syndactyly of third and fourth finger, clinodactyly of the fifth finger, syndactyly of second, third and fourth toes, microcephaly, broad base nose, severe physical retardation and intellectual disability. To our knowledge, 28 cases except ours have been described worldwide so far. All of the previously identified patients received a clinical diagnosis based on the finding of a typical phenotype, whereas all the genetic analyses performed such as karyotyping, Fluorescent In Situ Hybridization (FISH) analysis for the subtelomeric regions and Array-Comparative Genomic Hybridization (array-CGH), turned out to be negative. A single case report is available about a patient with Filippi-like syndrome harboring a 2q24.3q31.1 microdeletion that was identified by means of array-CGH analysis. Several case reports have been published of patients with a phenotype sharing some features with Filippi syndrome in which a 2q24q31 deletion was diagnosed. Recently in a Sardinian family with Filippi syndrome, Hussain et al performed genome-wide linkage analysis and identified several regions of homozygosity by descent including one on chromosome 2q13. In this region, wholeexome sequencing revealed a 1 bp insertion in exon 4 of the gene CKAP2L; Sanger sequencing confirmed the homozygous mutation and showed co-segregation with the disease phenotype in that family. CKAP2L encodes a polypeptide named cytoskeleton-associated protein-2-like or Radmis. It locates to the human centrosome and to the radial fibers and mitotic spindle of dividing neuronal stem cells of the murine embryonic and perinatal brain. In light of this recent discovery, after performing array-CGH, which did not reveal any microdeletions/microduplications, we proceeded with Sanger sequencing of all 9 CKAP2L exons including the adjacent splice sites in the child. This led to identification of a homozygous frameshift mutation, c.571dupA (p. Ile191Asnfs*6), which is the same mutation as previously described for the Sardinian family reported by Hussain et al—the first report on CKAP2L to be associated with Filippi syndrome. The identification of the homozygous mutation c.571dupA in the child described here confirmed our clinical suspicion and underlines the prominent role of CKAP2L mutations in Filippi syndrome. This diagnosis is helpful for risk assessment in the family. As this family and the one with the same mutation reported by Hussain et al are both of Sardinian extraction, it is tempting to speculate about a “founder effect” for this syndrome in the Sardinian population. If this will be corroborated by further Received: 6 October 2017 Revised: 27 November 2017 Accepted: 28 November 2017

Microduplication of the ARID1A gene causes intellectual disability with recognizable syndromic features

Purpose:To determine whether duplication of the ARID1A gene is responsible for a new recognizable syndrome.Methods:We describe four patients with a 1p36.11 microduplication involving ARID1A as identified by array–comparative genomic hybridization . We performed comparative transcriptomic analysis of patient-derived fibroblasts using RNA sequencing and evaluated the impact of ARID1A duplication on the cell cycle using fluorescence-activated cell sorting. Functional relationships between differentially expressed genes were investigated with ingenuity pathway analysis (IPA).Results:Combining the genomic data, we defined a small (122 kb), minimally critical region that overlaps the full ARID1A gene. The four patients shared a strikingly similar phenotype that included intellectual disability and microcephaly. Transcriptomic analysis revealed the deregulated expression of several genes previously linked to microcephaly and developmental disorders as well as the involvement of signaling pathways relevant to microcephaly, among which the polo-like kinase (PLK) pathway was especially notable. Cell-cycle analysis of patient-derived fibroblasts showed a significant increase in the proportion of cells in G1 phase at the expense of G2-M cells.Conclusion:Our study reports a new microduplication syndrome involving the ARID1A gene. This work is the first step in clarifying the pathophysiological mechanism that links changes in the gene dosage of ARID1A with intellectual disability and microcephaly.Genet Med advance online publication 01 December 2016