CUTTING EDGE TRIO-WGS IN RARE GENETIC SYNDROME DIAGNOSIS

INTRODUCTION

The implementation of high-throughput and deep sequencing methods in routine genetic diagnostics has significantly improved the diagnostic yield in patient cohorts with growth deficiencies and becomes increasingly more important as the prerequisite of personalised medicine. With the availability of genetic diagnostic laboratories with the latest technology, more and more patients should benefit from early diagnosis, specific targeted treatment, and better outcomes.

The cutting-edge genomic analysis techniques are revolutionizing the diagnosis of rare genetic diseases, allowing a complex analysis of the entire genome by identifying a wide spectrum of pathogenic allelic variants. Sequencing the entire exome/genome is increasingly becoming the first line test in situations where there is a high degree of uncertainty in the clinical diagnosis.

Persistent neonatal hypoglycemia can lead to severe neurodevelopmental consequences. Several congenital conditions should be considered in the diagnostic approach. Kabuki syndrome sometimes associates with neonatal hyperinsulinemic hypoglycemia. Kabuki syndrome is a rare neurodevelopmental disorder caused by variants in genes of histone modification, including KMT2D and KDM6A.

Among other manifestations encountered in KS are mentioned: craniofacial dysmorphism, microcephaly, hypertelorism, talipes valgus, hypotonia, joint hyperlaxity, global growth delay and superficial thrombophlebitis. The birth was premature, the newborn also needed assisted ventilation due to respiratory failure.

Current approaches to diagnosis of monogenic conditions include short-read sequencing of exomes or genomes Although the diagnostic yield from these methods is promising, ranging from 28 to 40% they leave many cases unresolved.

Patient clinical description and diagnostic algorithm

The patient, a 1-year-old female is referred to our genetics department. She presents a heterogeneous phenotype: facial dysmorphism, prematurity, respiratory failure requiring assisted ventilation, hypertelorism, superficial thrombophlebitis, microcephaly, talipes valgus, hypotonia, joint laxity and global development delay.

During the neonatal period, she had symptomatic hypoglycemia due to hyperinsulinism.

This required continuous glucose infusions to maintain normoglycemia in the first 3 days of life.

The first attempt to diagnose the patient’s syndrome was by using array-CGH, which yielded a normal molecular karyotype formula. Afterwards, the medical team decided that trio-WGS would be the optimal solution to diagnosing the disorder.

Genomic DNA was extracted from the EDTA blood sample using standard protocol. The library was prepared using TruSeq PCR-Free DNA Kit and sequencing was performed using NovaSeq 6000 (Illumina, San Diego, CA, USA). The result was reported using the databases Genome Reference Consortium Human Build 38 (GRCh38) and the Revised Cambridge Reference Sequence (rCRS) of the mitochondrial genome, generating an average genome-wide coverage of 42.56 readings.

Approximately 96.58% of the genome (98.93% of autosomes) was covered at >=20x coverage. In total, 4,182,355 single nucleotide variants (SNVs) and 1,019,245 small insertions and deletions (indels) were identified (1). Variants interpretation was performed using EVIDENCE (2) a software developed internally by 3billion Laboratory to prioritize variants based on the guidelines recommended by the American College of Genetic Medicine and Genomics (ACMG) and the Association for Molecular Pathology (AMP) (3,4) in the context of the patient’s phenotype, the relevant family history and the results of previous tests provided by the doctor recommending the analysis. Neonatal metabolic screening is under development for many metabolic and endocrine disorders (5).

Only variants considered clinically significant and relevant to the patient’s primary clinical indications at the time of interpretation of the variant will be reported. Confirmation of all SNVs, indels and SVs with breakpoints identified by Sanger sequencing was attempted.

A pathogenic, heterozygous, de novo variant, c.15535C>T was identified in the KMT2D gene. The genomic position: 12-49026431-G-A (GRCh38), protein: NP_003473.3:p.Arg5179Cys. This sequence change replaces arginine, which is basic and polar, with cysteine, which is neutral and slightly polar, at codon 5179 of the KMT2D protein (p.Arg5179Cys). This variant is not present in population databases (gnomAD no frequency). This missense variant has been observed in individual(s) with Kabuki syndrome (PMID: 27302555, 29536651). In at least one individual the variant was observed to be de novo. ClinVar contains an entry for this variant (Variation ID: 197586) (6).

The mode of transmission is autosomal dominant. The observed phenotype is that of Kabuki Syndrome 1 (OMIM#147920) (7-9).

Through trio-WGS sequencing, it was established that the only person with this variant is the newborn, and therefore a de novo variant.

DISCUSSION

The establishment of a precise molecular diagnosis can change the medical management, allow the estimation of a prognosis and complications’ risk, thus eliminating the uncertainty that affects the patient and her family.

As a result, trio sequencing is highly valuable for spotting pathogenic changes as well as for shedding light on the disease pathophysiology and inheritance patterns, such as de novo mutation (DNM) rates. It is noteworthy that some mutations discovered by trio sequencing may be “private” or rare variants that are specific to a single family. These families are constrained by the fact that both the genetic information and the clinical and biological phenotype are specific to one family.

Therefore, a thorough phenotypic functional characterization is required to confirm whether the disease is caused by this uncommon and one-of-a-kind mutation in order to deduce the function of such single (de novo) variations. Individuals with Kabuki syndrome have been shown to have this missense alteration. The variation has been observed to be de novo in at least one person.

Despite the remarkable diagnostic potential of genomic sequencing, there are challenges in the clinical application of the results. The challenges affect the laboratory (costs, optimal protocols, properly trained staff), the geneticist physician who interprets and reports the result, and the patient’s attending physician who must integrate the information obtained into the care plan. For this purpose, the phenotypic selection of patients eligible for such testing is very important and must be carried out following a detailed genetic consultation.

Although Kabuki syndrome can affect many systems and the disease is present from birth (congenital), it may take several years for symptoms to develop. The specific features and the severity of the patient’s condition may vary.

Neonatal hyperinsulinemic hypoglycemia (HH) is associated with Kabuki syndrome. The exact and sometimes overlapping molecular mechanisms leading to hypoglycemia in these syndromes have not been fully elucidated, although hyperinsulinism, augmented energy consumption, and dysregulation of growth hormone and cortisol have been reported. Early intervention is essential to minimize the risk of poor neurologic outcomes and developmental delay (11).

Table 1.

The most common features of Kabuki syndrome (10)

The most common features of Kabuki syndrome
Distinctive facial features	Abnormally long palpebral fissures with an abnormal curve on the outside edges Highly arched and broad eyebrows with sparse hair on the ends Short notch between nose and lips with a flat nose Large ears (that are cupped or sticking out)
Spinal abnormalities	Scoliosis or kyphosis
Fingers and toes abnormalities	Brachydactyly, clinodactyly Fleshy pads at the finger tips (persistent after birth)
Intellectual disability	Mild to moderate
Growth	Delay after birth

Patients with this syndrome also exhibit endocrine system-related conditions such as premature thelarche, precocious puberty, diabetes insipidus, thyroid dysfunction, obesity and growth hormone deficiency (12). In a recent study, Schott et al. discovered that 27.8% of patients with Kabuki syndrome sufferers with verified KMT2D mutations also exhibited growth hormone deficiency. In comparison to the has a 1% prevelance in the general population, growth hormone deficiency is common amongst Kabuki syndrome patients (13).

Some children may develop other signs and symptoms depending on the severity of their condition: feeding difficulties due to poor sucking and swallowing, malabsorption, gastroesophageal reflux, short stature, recurrent infections (such as ear infections, sinus infections or pneumonia), hearing loss, precocious puberty, kidney abnormalities, gastrointestinal abnormalities, immunological deficiencies, heart defects, persistently low blood sugar after birth (10,12).

Although it is challenging to define normal age ranges due to diversity when puberty begins in normal children, this definition can be used to describe precocious puberty before the age of 8 years in girls or 9 years in boys. Approximately 41% of patients with Kabuki syndrome present premature the larche, and some develop precocious puberty. These manifestations could be a result of reduced hypothalamus sensitivity to the suppressive effects of sex hormones on the release of gonadotropins (14).

The use of inheritance information which permits homozygosity mapping, inference of compound heterozygosity and the discovery of inheritance anomalies, makes trio sequencing superior to the sequencing of only one affected individual (15).

It is imperative and essential to adopt and implement common national and international standards to transform WGS from a research test into a reliable diagnostic test. The exchange of experience between the professionals involved in genomic sequencing is useful to compare the work protocol in the laboratory and to facilitate the common access to databases. Analysis interpretation and reporting of a WGS clinical test, in particular, is difficult due to the depth and magnitude of genetic variants that can be detected. Due to the comprehensive nature of the test, the laboratory must prioritize the detection of all variants relevant to the clinical phenotype, while minimizing reporting of highly uncertain or clinically irrelevant results. Currently, there is very little guidance or consensus from the ACMG or AMP regarding WGS testing, although there are a multitude of valid and justifiable strategies.

Conflict of interest

The authors declare that they have no conflict of interest.