Chromosome 4q duplication has been reported in over 60 cases (1), most as a result of chromosomal rearrangements which are inherited as unbalanced translocations from unaffected parents (2). Around half of these cases have been reported with a pure, tandem interstitial duplication, i.e., without other chromosomal anomalies, occurring directly after the original arrangement.

Phenotypic variation exists based on the exact location, size and expressivity of the duplicated genes (3). The reported phenotypic features include developmental delay (1, 4, 5), speech and language delay (6), psychomotor retardation (1, 7, 8), epilepsy or seizures (9) intellectual disability (1, 6, 10), microcephaly (3, 10, 11), vision problems (12, 13), growth retardation (4, 14), abnormalities to the extremities (14), retromicrognathia (3, 14, 15), deformed nasal bridge (16, 17), malformed ears (7, 14, 17) and other facial dysmorphisms (1, 11, 16, 18). Larger duplications are sometimes associated with renal or cardiac abnormalities (7, 18, 19). However, elucidating the causes of various phenotypes due to duplication in specific genes in the chromosomal region has been difficult due to the large number of genes affected and the phenotypic diversity.

We analysed a patient with a de novo ~38Mb (38,242,408bps) pure, direct interstitial 4q duplication. Pure duplications of this kind are important in establishing phenotype-gene correlations (20). Other types of 4q duplications involving translocations with other chromosomes are not reliable cases to establish such correlations as the new location could influence the resulting phenotype (21). Similarly, duplications involving interstitial inversions within 4q could also cause phenotypic changes, and hence unreliable for causal inference studies (22).

Considering the microduplication and the phenotypic characteristics in our patient, we attempted to elucidate the role those duplicated genes play in growth and development. We hypothesised that the phenotypes which the patient presented could be caused by duplications of the limited number of genes in the 4q region. Using a deductive reasoning approach through the retrospective analysis of previous case studies alongside the current case of pure partial 4q microduplication, we present the most likely casual genes which could be the critical dose sensitive genes in the 4q duplication syndrome.

A male patient presented as a premature child of 36 weeks at 2kg, delivered via c-section and admitted to the neonatal intensive care unit for oxygen supply. He had an uneventful gestational period and delivered to a healthy, non-consanguineous couple in their 20s (mother, gravida 3, para 3) and 30s (father), with no relevant medical history. The proband also has two healthy, unaffected siblings. The patient presented with microcephaly demonstrating a head circumference of below the 0.22^nd percentile (-3.57SDs). His height was below 1.45^th percentile (-2.19SDs) and his weight was at the 2.80^th percentile (-1.96 SDs) (Figures 1A–C). He exhibited some mild facial dysmorphism with a small face and receding chin.

At the last clinical visit, at the age of 5 years, the patient showed failure to thrive, aggressive behavioural issues and severe speech and language delay. His language capabilities were limited to three-word sentences incomprehensible to strangers. He did demonstrate comprehension for simple, familiar commands from his parents. He did not have any breathing difficulties; heart problems or other medical problem and his gross motor skills were within normal range.

To our knowledge, this is the first report of potential association of specific genes within the critical region of 4q28.1-32.1 with microcephaly, short stature and speech delay in 4q duplication syndrome. NAA15, the gene we identified as potentially associated with short stature from our phenotype-gene correlation analysis, is reported as a dosage sensitive gene on ClinGen, with reports of haploinsufficiency causing disease (24), but there are no reports on gene duplication. Heterozygous missense mutations in NAA15 were associated with speech, language delay, behavioural abnormality, intellectual disability, short stature, and poor growth (24, 25). NAA15 is a gene coding for an N-alpha-acetyltransferase 15 which forms a dimeric protein. This protein binds with NAA10 subunit to make a highly evolutionarily conserved Nat-A complex which is responsible for around 40% of post-translational acetylation modifications of human proteins. Extensive functional studies are lacking for the NAA15/Nat-A complex in humans so it is not certain how NAA15 could influence short stature or growth retardation. However, the Nat-A complex has been found to be crucial in embryogenesis and cellularization of endosperms in Arabidopsis (26–28) as well as in the early development of Danio Rario (29). Other studies have linked NAA10 with several cellular signalling pathways, including the JAK-STAT (30) and MAPK (31) pathways, which are known to be involved in GH-IGF-1 growth cascades. This suggests that NAA15 and the Nat-A complex are crucial in normal cell functioning (32) and that loss of function of this gene may decrease post-translational modifications on proteins involved in cellular processes leading to abnormal development.

Furthermore, it is not clear how overabundance of NAA15 could lead to pathogenesis. However according to one theory on the pathogenesis of CNVs affecting protein complex subunits; the overabundance of a single subunit from a complex may lead to a stoichiometric ratio imbalance leading to aberrant complex formation and or nonsense mediated decay (33–35). This could explain why we see a similar phenotype with both the loss and duplication of NAA15. The best way to establish this hypothesis would be to recapitulate the duplication in an in vitro cell line using patient-derived fibroblast cells or to recreate human pluripotent cells from the patient’s blood and to measure the level and size of the Nat-A complex being expressed compared to wild type controls.

PPV analysis supported GRIA2 as correlated with the speech delay phenotype in our patient. The gene itself lies 7.79Mbps away from the patient’s duplication breakpoint so it is unlikely to be disrupting the gene function. Mutations affecting GRIA2 are specifically responsible for causing neurodevelopmental disorders with language and behavioral abnormalities. Copy number variations of GRIA2 are generally rare, however it has been proposed that haploinsufficiency (deletions) of this gene may be pathogenic (36, 37). GRIA2 functions as a subunit for a glutamate cation channel receptor GluR2 in the brain and is activated by AMPA neurotransmitter binding; it plays a role in several neurological processes. Overexpression of GluR2 receptors could mean modification in Ca²⁺ permeability and conductivity of the ions (38). Mutations in the GRIA4, another receptor from the same GluR family, affect the gaining of long-term spatial reference memory (38). Similarly, duplication of GRIA2 could affect the dosage amount and levels of receptors available, negatively impacting the ability of individuals to learn and store language memory leading to speech delay.

PLK4 encodes for Polo Like Kinase 4, a protein that regulates centriole amplification during the cell division. The gene lies 0.872Mbps away from the patient’s duplication endpoint so it is unlikely to be disrupting the gene function. Pathogenic, homozygous frameshift and missense mutations in PLK4 often damage centriole biogenesis, microtubule spindle formation during mitosis, cell proliferation, viability and cause abnormal cytoskeleton formation (39–41). Specifically, these mutations often lead to increased cell mortality due to inefficient mitosis. Since PLK4 is pathogenic in a loss of function, autosomal recessive fashion, it is difficult to interpret how centriole biogenesis and mitosis could be compromised if the protein is duplicated and overexpressed.

Although renal and urinary system abnormalities were reported in previous cases, horseshoe kidneys seen in our patient were not shared by these probands. This suggests duplications of genes between 4q28.1-31.1 to be potentially associated with this phenotype.

The small number of patients with a duplication in this region is a limitation of the study; having a larger group of patients to compare to would strengthen the correlations derived from the analysis. Copy Number Variant (CNV) cases such as the one from this study are often recorded in the DECIPHER database. For this 4q region we found multiple patient cases with pure duplications in the database. However, the clinical information for the patients were insufficient, so they were not included in the present study. In the case of a similar study, the authors would contact the submitters for each of these cases and to obtain complete information for each patient. In addition, the possible effect of epistasis within this duplication region cannot be excluded at present, which may interfere with the attribution of a single gene per phenotype. Especially since some of the patients do not share the same phenotypes even though they share the same duplication region. Although this could also be due to incomplete penetrance or variable expressivity.

Further work is required to understand the exact mechanisms at play resulting from the triplosensitivity, since several mechanisms are known to responsible for leading to microcephaly, short stature and speech delay. In addition, the role of some the genes included in the duplication region remain to be elucidated; these could also be playing a role in the pathogenicity, but we cannot know unless further investigation is performed for those other genes. To validate our estimations on gene dosage pathogenicity, further experiments in cell and animal models to assess for phenotypic abnormalities are required. However, our study highlights the importance of analysing case reports with pure duplications in defining phenotype-gene relationships and in improving our knowledge of the function of precise chromosomal regions.

The patient’s father expressed his satisfaction at the current course of medical consultation. He was pleased that his son’s behavior with others and progress at school was improving. He noted how his son has become more social and has become clearer at speech. He did express his concern regarding his son’s surgical correction of hypospadias and noted that he was not happy that it was taking multiple surgeries for the repair to be completed. The patient’s father hopes that one day there will be treatment for genetic disorders such as his son’s.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Sidra Medicine. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the patient’s legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.

U-KU consented the family for publication, collected and analyzed the data and wrote the manuscript. KH supervised the clinical aspects of the study and contributed to writing. BH recruited the patient, collected blood samples and supported in writing. PJ supervised the work and wrote the manuscript. All the authors read and approved the final manuscript.

U-KU received PhD scholarship and PJ received research funds from the College of Health & Life Sciences, Hamad Bin Khalifa University, Qatar.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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