New study from China broadens the genetic picture of Kleefstra syndrome

A major new study from mainland China adds important information on the genetic and clinical diversity of Kleefstra Syndrome and highlights a particularly important issue for families, the parental mosaicism.

The work titled Genotype–phenotype correlations in 9q34.3 microdeletion syndrome: a study of 35 Mainland Chinese patients published in Orphanet Journal of Rare Diseases and included 35 new, previously unpublished KLEFS1 cases from mainland China. Specifically, the researchers studied 32 individuals and three fetuses with genetic findings consistent with KLEFS1.

The study identified 17 previously unreported variants of EHMT1, system , systematically examined the relationship between genotype and phenotype, presented data from the prenatal period, and recorded two cases in which the genetic alteration had been inherited from a mother with mosaicism.

Why a study from China was needed

Most of the largest cohorts of individuals with Kleefstra syndrome come from Europe, North America, or international registries.

Prior to this specific work, according to the authors, only ten individuals with KLEFS1 from mainland China.

Researchers collected the new cases through China League of EHMT1 Rare Disease between 2020 and 2024 and used genetic testing, medical records, and standardized questionnaires to create a more comprehensive picture of the syndrome in the specific population.

Adding data from diverse populations is important in rare diseases. The more and the more diverse individuals are studied, the better we can distinguish which characteristics are a core part of the syndrome and which may be associated with specific genetic variations.

35 new cases and 17 new variants of EHMT1

Overall, the researchers identified 21 different genetic alterations within the EHMT1 gene, of which 17 had not been previously described in the literature.

These changes were not all of the same type. They included:

• Nonsense variations, which prematurely create a «stop signal», resulting in the production of the protein being interrupted before completion.
• Frameshift variants, where the addition or removal of a small number of bases changes the way the DNA is «read» from that point on, usually resulting in a non-functional protein.
• Splice-site variants, which affect the proper processing of RNA before the protein is produced and can lead to an incorrect final product.
• Missense variants, in which a DNA «letter» changes and, as a result, one amino acid of the protein is replaced by another.

Simply put, it is different ways in which the normal function of EHMT1 can be disrupted. Other alterations prevent the production of the whole protein, while others change its structure or function.

From the recorded variants, 17 have not been previously reported in the literature.

At the same time, 14 individuals had deletions in the 9q34.3 region that included EHMT1. The size of the deletions varied enormously, from a very small deletion of just 120 bases affecting part of EHMT1 to a deletion of approximately 2.58 MB, which also included many neighboring genes.

Exactly this difference allowed researchers to examine one of the most important questions in Kleefstra.

What does EHMT1 itself cause, and what might the neighboring genes contribute?;

KLEFS1 can arise through different genetic pathways.

In some individuals, there is a pathogenic variant within the EHMT1 itself. In others, a segment of chromosome 9 is missing, including all or part of EHMT1. When the deletion is larger, other neighboring genes may also be missing at the same time.

The core question is therefore:

Which features are primarily caused by the loss of function of EHMT1, and which may become more frequent or severe because a larger deletion removes additional genes?;

To investigate this, the authors divided the participants into two main groups.

The first included 22 people with an EHMT1 variant or a deletion affecting only EHMT1.

The second included 9 people with a larger deletion that extended beyond EHMT1 and additionally included genes of the 9q34.3 region.

The main characteristics appear to be mainly due to EHMT1

One of the most important results was that for most of the key characteristics of KLEFS1 no statistically significant difference was found between the two genetic groups.

This concerned, among other things:

• developmental delay or intellectual disability,
• the neurological characteristics,
• behavioral problems,
• obesity,
• congenital heart defects and
• several other clinical manifestations.

Researchers believe that these results further support the view that EHMT1 haploinsufficiency, meaning the presence of only one functional copy of the gene is itself responsible for a large part of the core phenotype of Kleefstra Syndrome.

However, the largest deletions seem to add certain features

The picture was not the same for all features.

In individuals whose deletion included other genes in addition to EHMT1, the following were observed significantly more frequently:

• everted lower lip,
• small and sparse teeth,
• short neck and
• renal abnormalities.

For example, renal abnormalities had been recorded in 42,9% of the group with the largest deletions, versus 5,3% in the group where only EHMT1 was affected.

Similarly, the short neck was recorded at 66.7% compared to 13.3%, and the small, sparse teeth at 80% compared to 14.3%.

The authors consider that these differences support the possibility other genes or regulatory regions of 9q34.3 may contribute to certain additional features when included in a larger deletion.

However, this does not mean that we can accurately predict the clinical picture of a specific child solely from the size of the deletion.

The individual study groups were small and the results need confirmation in larger populations.

A greater deletion did not necessarily mean greater developmental delay

This is a particularly interesting point of the study.

In 16 children A standardized evaluation was performed using the Gesell Developmental Schedules, examining:

• gross motor skills,
• fine motor skills,
• Language,
• personal and social skills and
• adaptive behavior.

The researchers they did not find a significant difference in the scores between the children with isolated EHMT1 loss and those with a larger deletion that also included additional genes.

Language development was particularly affected in both groups.

The finding is significant because previous studies had suggested that larger deletions may generally be associated with more severe intellectual disability.

In this specific Chinese team, this was not confirmed when growth was measured with a standardized tool.

Again, the participants were too few for the issue to be considered definitively resolved.

What were the most frequent characteristics of the group

For the main clinical analysis, data from were used 31 people, since the three embryos were not included and for one more person there was insufficient clinical data.

Developmental delay or intellectual disability had been recorded in 31 out of 31 people.

Speech delay and motor delay were present in 96,7%.

Other common features were:

• MRI findings in the 50% group among those for whom an MRI was available,
• a decline to 46.2%,
• stereotypical behaviors at 46.2%,
• difficulty chewing at 43.5%,
• gait abnormalities in 40%,
• associated cardiac abnormalities in 38.5% and
• constipation at 37.5%.

The average age of independent walking acquisition was approximately 32 months, while in the latest case independent walking was achieved at the age of six years.

These percentages describe this specific group and should not be considered accurate estimates for all individuals with Kleefstra.

For the first time, more data than even from pregnancy

Another interesting aspect of the work was the retrospective recording prenatal data.

Pregnancy information was available for 26 cases, including the three fetuses.

In 42,3% Prenatal ultrasounds had been described as normal.

The most common abnormal finding was small for gestational age, which was recorded in 15.41 TP3T. Findings involving the brain, small head circumference, polyhydramnios, cardiac or renal abnormalities, and other congenital findings were also reported, though in smaller numbers.

These data are interesting, but they do not specify a particular prenatal ultrasound pattern for Kleefstra.

Many of the findings are non-specific and, as the study itself shows, in a significant percentage of pregnancies the ultrasound was normal.

Therefore, the diagnosis remains genetic and cannot be made or ruled out by a normal ultrasound.

A particularly significant finding, parental mosaicism

Perhaps the finding of greatest immediate significance for genetic counseling concerns two families.

In most KLEFS1 cases, the pathogenic variant is considered again, meaning it appears for the first time in the child and is not detected as a normal heterozygous variant in either parent.

However, in this specific study, Of the 28 probands for whom parental origin could be assessed, approximately 7% had inherited the EHMT1 variant from a mother with mosaicism.

The 7% applies exclusively to this small series and the true frequency of parental mosaicism in Kleefstra should not be considered.

Nevertheless, the finding is very important.

What is parental mosaicism

Mosaicism means that the genetic alteration it is not present in all parent cells, but only in a percentage of them.

A parent may therefore not have the typical appearance of Kleefstra syndrome or may present only very mild features because a large portion of their cells possesses normal EHMT1.

In one of the two families in the study, the mother had a mosaic variant c.3310G>A. It did not present a typical KLEFS1 picture, although the researchers described certain mild morphological features and obesity.

In the second family, another variant of EHMT1 was found in the mother in mosaic form, with approximately 27% of the readings in this specific examination, bring the change.

Why mosaicism matters for recurrence risk

When a variant has truly arisen de novo in the child, the risk of it occurring again in a subsequent pregnancy is usually considered low, although it is not completely zero due to the possibility of gonadal mosaicism.

However, if a parent has proven mosaicism, the situation changes.

Genetic alteration may also exist in a portion of the oocytes or spermatozoa and therefore the risk of transmission may be greater than that of a purely de novo case.

However, a single percentage cannot be calculated solely from the ratio of blood variation, because the degree of mosaicism may differ in the reproductive tissues.

For this reason, the identification of mosaicism has immediate significance for personalized genetic counseling and family planning.

A negative test in the parent does not always answer everything

Mosaicism also highlights a broader issue.

A genetic test is usually performed on blood. However, if a mutation is present in a very small percentage of cells or mainly in a different tissue, it can be more difficult to detect.

This is one of the reasons why genetic counseling after a KLEFS1 diagnosis remains important even when the variant initially appears de novo.

This specific work does not mean that all parents must be considered mosaic. It does show, however, that parental mosaicism is a real possibility and not just a theoretical exception.

We shouldn't generalize the 7%

The finding of the two families is impressive, but it requires special attention.

The two cases correspond to 2 of 28 probands in this specific Chinese series.

When the total number is so small, even two incidents create a relatively large percentage.

We cannot, therefore, say that «the 7% in families with Kleefstra has parental mosaicism.».

Much larger series, as well as sensitive genetic techniques, are needed to estimate the true frequency.

The message is different:

Mosaicism must be seriously considered when evaluating the origin of an EHMT1 variant and the risk of recurrence in a family.