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breast, she was fed on infant formula. The first neuropediatric assessment occurred at three months of
age, showing OFC parameters of 35.2 cm (-3 SD), weight of 4,600 g (25th percentile), and length of 58
cm (50th-75th percentile). At the same age, cerebral ultrasound gave normal results. At last evaluation
at the age of 8 months, her OFC was 39 cm (-3 SD) and weight was 7.5 kg (10th-25th). Minor facial
dysmorphisms were noted [Figure 1D]. CMA analysis, performed at birth in light of her father’s CMA
finding, highlighted the same 1q24.3q25.2 deletion of 5.8 Mb [Supplementary Figure 1D]. At the age of 41 days,
routine chromogenic plasma testing revealed low antithrombin activity 3 (32%, normal 80%-120%), similar
[3]
to what was documented in the father (45%, normal 70%-130%) .

Molecular investigations
After obtaining the informed consent approved by the ethics committee for research at the corresponding
institutions, DNA samples were prepared from blood of all six subjects and their parents. The study was
conducted in accordance with the Declaration of Helsinki and national guidelines.

Chromosomal microarray analysis
CMA was performed on all six subjects and their parents using Agilent Human Genome CGH Microarray
Kit 4 × 180 k (G4449A) and CGH-SNP array (G4890A) in order to investigate the parental origin of the
CNV and whether it was de novo or inherited.

Data analysis was done using Agilent Cytogenomics V.2.5.8.1. All nucleotide and SNP positions refer to the
Human Genome, February 2009 Assembly (hg19). Molecular karyotype was designed according with the
International System for Human Cytogenetic Nomenclature (ISCN 2016).

Parent of origin determination
We genotyped family trios in three subjects (Cases 2, 3, and 6) by CGH-SNP array (Agilent 4 × 180k).

Gene content analysis
The gene content for each SRO was analyzed taking into account the haploinsufficiency (HI) and loss-of-
function intolerance (pLI) scores. The HI score is defined as the predicted probability that a gene is more
likely to exhibit haploinsufficiency (0%-10%) or more likely not to exhibit haploinsufficiency (90%-100%)
based on differences in characteristics between known haploinsufficient and haplosufficient genes (https://
decipher.sanger.ac.uk/).

The pLI score represents the probability that a gene is extremely intolerant of loss-of-function variation
(pLI ≥ 0.9). Genes with low pLI scores (≤ 0.1) are loss-of-function tolerant. This score is based on protein-
truncating variants in the GnomAD database (https://gnomad.broadinstitute.org/). Moreover, according
to gnomAD Gene constraint suggestions, to evaluate highly likely haploinsufficient genes, we also used the
observed/expected score.

Whole-exome sequencing analysis
Whole-blood samples of all available family members [Figure 3], except for the newborn baby (Case 6),
were collected for WES analysis, which was performed by an external service provider (BGI Genomics,
Hong Kong). According to the provider’s description, whole-exome enrichment was carried out using
Illumina kit and sequenced with the DNB-SEQ500 to generate 100-bp-paired end reads that were aligned
to the human genome (UCSC GRCh38), at an average coverage of 150 ×.

[11]
GATK tools (https://software.broadinstitute.org/gatk/) , including base quality score recalibration,
indel realignment, duplicate removal, and SNP, and insertions/deletion (INDEL) identification were
[12]
used according to the recommendations of GATK best practices . Variants were annotated using the