Background: Among aneuploidies compatible with life, trisomy 22 mosaicism is extremely rare, and only about 25 postnatal and 18 prenatal cases have been described in the literature so far. The condition is mainly characterized by facial and body asymmetry, cardiac heart defects, facial dysmorphisms, growth failure, delayed puberty, and variable degrees of neurodevelopmental delay. Problem: The scattered information regarding the condition and the dearth of data on its natural history and developmental outcomes restrict genetic counseling, particularly in prenatal settings. Moreover, a prompt diagnosis is frequently delayed by the negative selection of trisomic cells in blood, with mosaicism percentage varying among tissues, which often entails the need for further testing. Purpose/topic: The aim of our work is to provide assistance in prenatal and postnatal genetic counseling by systematically delineating the current knowledge of the condition. This entails defining the prenatal and postnatal characteristics of the condition and presenting novel data from three cases, both prenatally and postnatally. Additionally, we report the developmental outcomes observed in two new patients.

(1) Background: The aim of our study is to evaluate whether cell-free DNA testing can overlap the genetic testing of miscarriage tissue in women with early pregnancy loss (EPL) and length of recurrent pregnancy loss (RPL); (2) Methods: We conducted a prospective cohort study at the Pregnancy Loss Unit of the Fondazione Policlinico Universitario A. Gemelli (IRCCS), Rome, Italy between May 2021 and March 2022. We included women with EPL and length of RPL. Gestational age was >9 weeks + 2 days and 25 and <54 mm. Women underwent both dilation and curettage for the collection of miscarriage tissue and for blood sample collection. Chromosomal microarray analysis (CMA) on miscarriage tissues was performed by oligo-nucleotide- and single nucleotide polymorphisms (SNP)-based comparative genomic hybridization (CGH+SNP). Maternal blood samples were analyzed by Illumina VeriSeq non-invasive prenatal testing (NIPT) to evaluate the cell-free fetal DNA (cfDNA) and the corresponding fetal fraction and the presence of genetic abnormalities; (3) Results: CMA on miscarriage tissues revealed chromosome aneuploidies in 6/10 cases (60%), consisting of trisomy 21 (5 cases) and monosomy X (one case). cfDNA analysis was able to identify all cases of trisomy 21. It failed to detect monosomy X. A large 7p14.1p12.2 deletion concomitant to trisomy 21 was, in one case, detected by cfDNA analysis but it was not confirmed by CMA on miscarriage tissue. (4) Conclusions: cfDNA largely reproduces the chromosomal abnormalities underlying spontaneous miscarriages. However, diagnostic sensitivity of cfDNA analysis is lower with respect to the CMA of miscarriage tissues. In considering the limitations when obtaining biological samples from aborted fetuses suitable for CMA or standard chromosome analysis, cfDNA analysis is a useful, although not exhaustive, tool for the chromosome diagnosis of both early and recurrent pregnancy loss.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism caused by the instability of a CGG trinucleotide repeat in exon 1 of the FMR1 gene. The co-occurrence of FXS with other genetic disorders has only been occasionally reported. Here, we describe three independent cases of FXS co-segregation with three different genetic conditions, consisting of Duchenne muscular dystrophy (DMD), PPP2R5D--related neurodevelopmental disorder, and 2p25.3 deletion. The co-occurrence of DMD and FXS has been reported only once in a young boy, while in an independent family two affected boys were described, the elder diagnosed with FXS and the younger with DMD. This represents the second case in which both conditions coexist in a 5-year-old boy, inherited from his heterozygous mother. The next double diagnosis had never been reported before: through exome sequencing, a girl with FXS who was of 7 years of age with macrocephaly and severe psychomotor delay was found to carry a de novo variant in the PPP2R5D gene. Finally, a maternally inherited 2p25.3 deletion associated with a decreased level of the MYT1L transcript, only in the patient, was observed in a 33-year-old FXS male with severe seizures compared to his mother and two sex- and age-matched controls. All of these patients represent very rare instances of genetic conditions with clinical features that can be modified by FXS and vice versa.

Neuronal ceroid lipofuscinoses (CNL) are lysosomal storage diseases that represent the most common cause of dementia in children. To date, 13 autosomal recessive (AR) and 1 autosomal dominant (AD) gene have been characterized. Biallelic variants in MFSD8 cause CLN7 type, with nearly 50 pathogenic variants, mainly truncating and missense, reported so far. Splice site variants require functional validation. We detected a novel homozygous non-canonical splice-site variant in MFSD8 in a 5-year-old girl who presented with progressive neurocognitive impairment and microcephaly. The diagnostic procedure was elicited by clinical genetics first, and then confirmed by cDNA sequencing and brain imaging. Inferred by the common geographic origin of the parents, an autosomal recessive inheritance was hypothesized, and SNP-array was performed as the first-line genetic test. Only three AR genes lying within the observed 24 Mb regions of homozygosity were consistent with the clinical phenotype, including EXOSC9, SPATA5 and MFSD8. The cerebral and cerebellar atrophy detected in the meantime by MRI, along with the suspicion of accumulation of ceroid lipopigment in neurons, prompted us to perform targeted MFSD8 sequencing. Following the detection of a splice site variant of uncertain significance, skipping of exon 8 was demonstrated by cDNA sequencing, and the variant was redefined as pathogenic.

Background: Macrocephaly can be a component manifestation of several monogenic conditions, in association with intellectual disability/developmental delay (ID/DD) behaviour abnormalities, including autism spectrum disorders (ASD), and variable additional features. On the other hand, idiopathic ASD can present with developmental delay and macrocephaly. Methods: We carried out a retrospective analysis of a cohort of 78 patients who were tested from February 2017 to December 2024 by high-throughput sequencing of a panel of 27 genes (ABCC9, AKT1, AKT2, AKT3, BRWD3, DIS3L2, DNMT3A, EZH2, GPC3, GPC4, HERC1, MED12, MTOR, NFIA, NFIX, NSD1, PDGFRB, PIK3CA, PIK3R1, PIK3R2, PPP2R1A, PPP2R5D, PTEN, RAB39B, RNF135, SETD2, and TBC1D7) because of neurodevelopmental impairment, including ID/DD, ASD/behaviour abnormalities associated with macrocephaly, mimicking to a large extent idiopathic ASD. Results: Pathogenic variants leading to the diagnosis of monogenic conditions were detected in 22 patients (28%), including NSD1 (2), PTEN (16), and PPP2R5D (4). Distinctive of the PTEN-associated phenotype were true macrocephaly (100%), ASD or behaviour abnormalities (92%), mild/borderline ID (79%), and no facial dysmorphisms. Typical of the PPP2R5D-associated phenotype were relative macrocephaly (75%), a few unspecific peculiar facial characteristics (50%), and a more variable presentation of the neurodevelopmental phenotype. Conclusions: Pathogenic variants in PTEN and PPP2R5D are the most recurrent gene mutations in a patient-oriented procedure for the genetic diagnosis of apparently idiopathic ASD and behaviour abnormalities associated with macrocephaly. The clinical applicability of the presented diagnostic strategy is discussed.

We describe the complex case of a 44-year-old man with polycystic kidney disease, mild cognitive impairment, and tremors in the upper limbs. Brain MRI showed lesions compatible with leukodystrophy. The diagnostic process, which included clinical exome sequencing (CES) and chromosomal microarray analysis (CMA), revealed a triple diagnosis: autosomal dominant polycystic kidney disease (ADPKD) due to a pathogenic variant, c.2152C>T-p.(Gln718Ter), in the PKD1 gene; late-onset phenylketonuria due to the presence of two missense variants, c.842C>T-p.(Pro281Leu) and c.143T>C-p.(Leu48Ser) in the PAH gene; and a 915 Kb duplication on chromosome 15. Few patients with multiple concurrent genetic diagnoses are reported in the literature; in this ADPKD patient, genome-wide analysis allowed for the diagnosis of adult-onset phenylketonuria (which would have otherwise gone unnoticed) and a 15q11.2 duplication responsible for cognitive and behavioral impairment with incomplete penetrance. This case underlines the importance of clinical genetics for interpreting complex results obtained by genome-wide techniques, and for diagnosing concurrent late-onset monogenic conditions.