Homozygous 22q11.2 distal type II microdeletion is associated with syndromic neurodevelopmental delay

Genomic disorders result from heterozygous copy number variants (CNVs). Homozygous deletions spanning numerous genes are rare, despite the potential contribution of consanguinity to such instances. CNVs in the 22q11.2 region are mediated by nonallelic homologous recombination between pairs of low copy repeats (LCRs), from amongst eight LCRs designated A‐H. Heterozygous distal type II deletions (LCR‐E to LCR‐F) have incomplete penetrance and variable expressivity, and can lead to neurodevelopmental issues, minor craniofacial anomalies, and congenital abnormalities. We report siblings with global developmental delay, hypotonia, minor craniofacial anomalies, ocular abnormalities, and minor skeletal issues, in whom chromosomal microarray identified a homozygous distal type II deletion. The deletion was brought to homozygosity as a result of a consanguineous marriage between two heterozygous carriers of the deletion. The phenotype of the children was strikingly more severe and complex than that of the parents. This report suggests that the distal type II deletion harbors a dosage‐sensitive gene or regulatory element, which leads to a more severe phenotype when deleted on both chromosomes.

Homozygous deletions may result from regions of homozygosity (ROH) due to identity-by-descent in consanguineous populations, which would be expected to also result in homozygosity of larger CNVs.Alternatively, nonallelic homologous recombination (NAHR)mediated recurrent deletions may have arisen de novo in recent generations, as the mutation rate of NAHR at a given locus can be as high as $10 À4 to 10 À5 (Turner et al., 2008;Yuan et al., 2022).Nonetheless, the relative lack of larger homozygous deletions involving multiple genes in the literature is striking.Reasons for this may include pathogenicity of the heterozygous CNV with reduced reproduction rates, or embryonic lethality when multiple genes are deleted.Examples of such rarely reported homozygous deletions involving multiple genes to include the recurrent NAHR-mediated deletion including NPHP1 [MIM 607100] leading to nephronophthisis or Joubert syndrome (Parisi et al., 2004;Saunier et al., 2000), a homozygous deletion in 15q24.1 including CYP1A1 and EDC3 (Zhang et al., 2021), and a homozygous deletion of 21q22.2 in a child with global developmental delay, hypotonia, cortical visual impairment, and mild craniofacial anomalies (Hildebrandt et al., 2021).
We report siblings with global developmental delay, hypotonia, mild craniofacial anomalies, and ocular anomalies, in whom CMA revealed a homozygous 22q11.2distal deletion.The phenotype of both children was more severe than that of the parents and previously reported heterozygous cases, providing further confirmation of a dosage effect for this region.

| Subjects
The family provided written consent for publication.

| Chromosomal microarray analysis
Both affected siblings and the parents had a clinical CMA (Affymetrix CytoScan 750K array), with a resolution level of 50 kb (25 probes).
Analysis was done on the Affymetrix Chromosome Analysis Suite 3.1 (genome build 37, GRCh37).

| Exome sequencing
Following informed consent, exome analysis was performed on DNA extracted from whole blood of individual III-2.Exonic sequences from DNA were enriched with the xGen Exome Research Panel IDT-V2.
Sequences were generated on a NovaSeq 6000 system (Illumina, San Diego, CA).Read alignment and variant calling were performed with DNAnexus (Palo Alto, CA) using default parameters with the human genome assembly hg19 (GRCh37) as reference.Exome analysis of the proband yielded 45 million reads, with a mean coverage of 64Â.Following alignment to the reference genome [hg19] and variant calling, variants were filtered out if they were off-target (>8 bp from splice junction), synonymous, or had minor allele frequency >1% in the gno-mAD database.

| Clinical report
The two affected siblings (Figure 1a, Individuals III-1 and III-2; Table 1) were born to consanguineous (second cousins once removed) parents of Muslim-Arab origin.Individual III-1 was a female born at full term by Caesarean section, at a birthweight of 2700 g.The pregnancy was complicated by maternal gestational diabetes, controlled by dietary management.After birth, she had mild hypotonia and motor delay, with walking at around age 2 years of age.At 5 years of age, she walked with a tip-toeing gait and had recurrent falls and difficulty climbing stairs.In addition, she had cognitive delay, speech delay (sentences of two words with mostly incomprehensible speech), behavioral problems, and hyperactivity.She had recurrent episodes of wheezing, and her work-up showed slightly elevated IgE (68.8 IU/mL, normal 0-52 IU/mL), with otherwise normal immunoglobulin levels and IgG subclasses.Ophthalmic exams, initially at 12 months, showed myopia and strabismus.Additional evaluations included normal hearing exam, normal creatine phosphokinase (CPK) level at 11 months, and a small patent foramen ovale (PFO) on echocardiogram in infancy.
On clinical evaluation at 5 years of age, growth parameters were within normal range: head circumference was 52 cm (88th percentile), height measured 107 cm (44th percentile), and weight was 23 kg (92nd percentile).Facial features included round face, low-set ears, and esotropia.She had one hyperpigmented macule on the right side of the face, and flat feet.
The second affected sibling (Figure 1a, Individual III-2) was a male born at full term by Caesarean section after an uneventful pregnancy, at a birthweight of 2400 g.Since an early age, he had torticollis with hypotonia and received physical therapy.Developmental milestones were delayed; he started to walk at around 3 years of age, and at 3 years 7 months had recurrent falls with difficulties climbing stairs, and he could only say a few words.He had cognitive delay, stereotyped movements, behavioral problems, and hyperactivity.CPK was normal at 17 months.He had recurrent acute otitis media as well as serous otitis media, and hearing exam showed conductive hearing impairment.Ophthalmic evaluation at 16 months showed a flat right optic disc with peripapillary atrophy and scar below the disc.Brain MRI revealed prolonged relaxation times in the left parietal periventricular area.Abdominal and renal ultrasound at 2.5 years were normal.
At least eight probands with heterozygous distal type II deletions and detailed clinical investigations were previously reported in the literature (Rauch et al., 2005;Shaikh et al., 2000Shaikh et al., , 2007)), as well as three prenatal cases (Burnside, 2015) and numerous submissions to DECI-PHER (https://www.deciphergenomics.org/).The clinical and T A B L E 1 (Continued)  (Burnside, 2015) and in a larger, heterozygous distal deletion (Rauch et al., 2005), and may be secondary to recurrent infections although this was not reported in the current case.The phenotype of the individuals in this report is strikingly more severe than that of either parent, in line with the expected effect of dosagesensitive genes within this interval.This is consistent with reports of triplications being more severe than pathogenic duplications (Liu et al., 2014).Alternatively, there may be modifiers acting in trans to the heterozygous deletions, which dictate penetrance and expression.
The distal type II microdeletion includes five protein-coding genes (IGLL5, RSPH14, GNAZ, RAB36, and BCR) and a microRNA (mir-5571), yet the dosage-sensitive gene in this interval has not yet been identified.Aside from IGLL5, the protein-coding genes are all expressed in brain (GTEx portal, www.gtexportal.org).Two of the genes have constraint metrics compatible with haploinsufficiency, as indicated by a high probability of being loss-of-function intolerant (pLI) score and low observed/expected (o/e) ratio per gnomAD (Lek et al., 2016)-GNAZ (pLI 0.96; o/e 0.29) and BCR (pLI 0.99; o/e 0.17).BCR has been well characterized, as it is the site of the breakpoint in the Philadelphia chromosome translocation found in chronic myeloid leukemia (CML) and acute lymphocytic leukemia, resulting in the chimeric oncogene BCR-ABL (Laurent et al., 2001).RAB36, encoding RAS-associated protein RAB3B [MIM 605662] is an interesting candidate for neurodevelopmental disorders, although its pLI score is 0 and the o/e ratio is 0.83.It is important for neurite outgrowth through regulation of endocytic recycling downstream of RAB35 and MICALL1, in response to nerve growth factor (Kobayashi et al., 2014).Alternatively, this region may harbor a regulatory element influencing a gene not included within the E-F interval.
In summary, we present two cases with a homozygous distal type II microdeletion and a review of the literature.Our data suggest that there is a dosage-sensitive gene or regulatory element within this interval, and that the homozygous deletion leads to a more severe, syndromic neurodevelopmental phenotype.

F
I G U R E 1 Pedigree and CMA results.(a) Pedigree indicating the two affected siblings and parental consanguinity.Both parents are heterozygous for the 22q11.2distal type II deletion.Light gray shapes indicate individuals with cystinosis, unrelated to the 22q11.2deletion.(b) Schematic drawing of 22q11.2region, indicating LCRs A-H, based on UCSC genome browser [GRCh37/hg19].(c) Homozygous deletion of LCRs E-F in the affected child (upper panel) and heterozygous deletions in both parents (middle and lower panels).T A B L E 1 Clinical features and review of reported cases.Rauch et al., 2005 Shaikh et al.

Family
history was positive for learning difficulties in the father, who completed only 7-8 years of elementary school, and for children with learning difficulties in the extended family.The mother reportedly had normal development and cognition, and had 2 years of university training.The younger sibling was an infant, and did not yet undergo cytogenetic testing.In addition, several members of the extended family had an unrelated diagnosis of cystinosis due to a missense variant in CTNS.3.2 | Homozygous deletion detected on chromosomal microarrayClinical CMA of both affected siblings demonstrated a shared homozygous 650 kbp deletion in chromosome 22q11.2,arr[GRCh37] 22q11.22q11.23(22997928_23652519)x0,corresponding to the distal microdeletion type II, which is mediated by low copy repeats (LCRs) E-F (Figure1b,c).Both parents were heterozygous for the deletion.In order to exclude additional monogenic causes contributing to the clinical manifestations, exome sequencing was pursued for the second affected sibling (individual III-2) and no additional pathogenic or likely pathogenic variants were detected in genes associated with the phenotype.4| DISCUSSIONChromosome 22q11.2contains a cluster of eight LCRs designated LCR A-H, which predispose the region to deletions and duplications mediated by meiotic NAHR(Shaikh et al., 2007).CNVs in this region are classified into proximal, central, and distal rearrangements.Of these, the most common is the $3 Mb deletion mediated by LCR-A and LCR-D, leading to the proximal 22q11.2deletion syndrome (DiGeorge [MIM 188400] and velocardiofacial[MIM 192430] syndromes).Central deletions are nested at the distal end of the proximal $3 Mb interval, but do not include the dosage-sensitive genes TBX1 level of sequence variation, correlated with a lower frequency of NAHR-mediated CNVs.Distal deletions are sub-divided into three types: type I (LCR-D to LCR-E/F), type II (LCR-E to LCR-F), and type III (minimal region-LCR-F to LCR-G and encompassing SMARCB1; features of the eight cases with detailed clinical information and of the present cases are summarized in Table1.In four individuals, the deletions were reported to occur de novo.Two of the reported individuals, as well as the father reported inRauch et al. molecular