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\begin{document}

\title{Report of Two cases of Schaaf-Yang Syndrome: Same Genotype, Different
Phenotype}



\author[1]{Ana Maria Rodriguez Barreto}%
\author[1]{Katherine  Schain}%
\author[1]{Parul Jayakar}%
\author[2]{Meredith  Wright}%
\author[2]{Shimul  Chowdhury}%
\author[1]{Daria  Salyakina}%
\affil[1]{Nicklaus Children's Hospital}%
\affil[2]{Rady Children's Institute for Genomic Medicine}%


\vspace{-1em}



  
  \date{May 20, 2024}


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\sloppy


Report of Two cases of Schaaf-Yang Syndrome: Same Genotype, Different
Phenotype

Ana Maria Rodriguez,\textsuperscript{a} MD, Katherine
Schain,\textsuperscript{a} MS, CGC, Parul
Jayakar,\textsuperscript{a}MD,MS,FACMG, Meredith S.
Wright,\textsuperscript{b,c} PhD., Shimul Chowdhury,\textsuperscript{b}
PhD., Daria Salyakina,\textsuperscript{e}PhD.

\textsuperscript{a} Division of Genetics and Metabolism, Nicklaus
Children's Hospital Pediatric Specialists, Miami, (FL,)
USA.\textsuperscript{b} Rady Children's Institute for Genomic Medicine,
San Diego, (CA,) USA. \textsuperscript{c} Keck Graduate Institute,
Claremont, (CA,) USA. \textsuperscript{d} Personalized Medicine \&
Health Outcomes Research, Nicklaus Children's Hospital Pediatric
Specialists, Miami, (FL,) USA

\textbf{Corresponding Author:} Ana Maria Rodriguez Barreto

Division of Genetics and Metabolism, Nicklaus Children's Hospital
Pediatric Specialists, Miami, (FL,). 3100 SW 62nd Avenue, Miami,
FL,~33155, USA. Tel: 786-624-4717. Fax : 786-624-4704. E-mail:
anamaria.rodriguezbarreto@nicklaushealth.org

\textbf{Co-authors:} Katherine Schain:
Katherine.Schain@Nicklaushealth.org

Parul Jayakar: Parul.Jayakar@Nicklaushealth.org

Meredith S. Wright: MWright3@rchsd.org

Shimul Chowdhury: schowdhury@bluestargenomics.com

Daria Salyakina: Daria.Salyakina@Nicklaushealth.org

\textbf{Conflicts of Interests Statement:} We know of no conflicts of
interests associated with this publication, and there has been no
significant financial support for this work that could have influenced
its outcome. As Corresponding Author, I confirm that the manuscript has
been read and approved for submission by all the named authors.

\textbf{Informed Consent} : Written informed consent was obtained from
both patients parents.

\textbf{Ethics Declaration:} The study was conducted according to the
guidelines of the Declaration of Helsinki and approved by The
Institutional Review Board (or Ethics Committee) of Nicklaus Children's
Hospital Pediatric Specialists.

\textbf{KEY CLINICAL MESSAGE}

We report two, genotypically identical but phenotypically distinct cases
of Schaaf-Yang Syndrome and propose the early use of Genome Sequencing
in patients with unspecific presentations to facilitate the early
diagnosis of children with rare diseases.

\textbf{INTRODUCTION}

Rare diseases are defined as any disease or condition that affects less
than 200,000 people in the United States\textsuperscript{1}.~Because of
their rarity, these conditions are difficult to diagnose and therefore,
the time to diagnosis is prolonged and often involves extensive and
expensive work up (i.e. the diagnostic odyssey). With a prevalence of
\textless{} 1/1,000,000\textsuperscript{2}, Schaaf-Yang Syndrome (SYS,
OMIM \#615547) caused by pathogenic variants in the MAGEL2 gene, is a
rare disease with a very unspecific phenotype which overlaps with
numerous other more common conditions, hence, defaulting it's diagnosis
even further. Nonetheless, with the advent of Next Generation Sequencing
(NGS) and with it, broad sequencing techniques, more individuals with
SYS have been identified and some phenotypic-genotypic associations have
been proposed\textsuperscript{3}. However, diagnosis continues to be
delayed and achieved only after extensive work up has been done, without
significant impact in management once diagnosis has been achieved. The
rapid Whole Genome Sequencing (rWGS) protocol was developed at Nicklaus
Children's Hospital to facilitate the early diagnosis of children with
rare genetic conditions to guide management strategies and improve
long-term outcomes. We report two, genotypically identical but
phenotypically distinct, cases of SYS diagnosed through the rWGS
protocol and propose the early utilization of WGS in patients with
poorly specific presentations to facilitate the diagnosis of patients
with rare genetic diseases. In turn, this will allow for better
understanding of these conditions and guide targeted management
approaches that can positively influence health care outcomes in the
acute and long term settings.

\textbf{Keywords:} MAGEL2, rapid Whole Genome Sequencing,
phenotype-genotype association, ICU

\textbf{CASE PRESENTATION}

\emph{Patient 1}

Full Term male transferred to Nicklaus Children's Hospital's neonatal
intensive care unit (NICU) at one week of age for concerns of an
underlying genetic etiology given dysmorphic features, respiratory
insufficiency and feeding difficulties. He was born via C-section from a
pregnancy complicated by intrauterine growth restriction (IUGR) and
clubfeet identified on second trimester ultrasound. At birth, he was
noted to be small for gestational age (SGA) (birth weight near
2\textsuperscript{nd} percentile) and to have a wide posterior
fontanelle, down slanting palpebral fissures, micrognathia with limited
mouth opening; bilateral cryptorchidism; arthrogryposis multiplex
congenita (flexion deformity of wrists and proximal metacarpophalangeal
joints and bilateral talipes equinovarus); bilateral club feet;
Mongolian spot on the right buttock; hypertonia of upper and lower
extremities; weak suck and gag reflexes and; absent rooting and Moro
reflexes. Perinatal course was complicated by feeding difficulties with
nasogastric tube (NG tube) dependence, respiratory distress requiring
invasive respiratory support, apneic episodes and hypoglycemia requiring
treatment with dioxide.

Chest and abdominal x-rays, abdominal ultrasound, and echocardiogram
were reported as normal. Bone survey demonstrated findings consistent
with arthrogryposis, bilateral clubfeet, and eleven ribs. CT scan of the
face revealed a hypoplastic mandible, flattening of the condylar heads,
and bilaterally shallow temporomandibular joint glenoid fossa.

Botox injection was performed to help manage oral secretions at 1 month
of age. Subperiosteal release of the mouth floor was performed at 1
month of age to help with oral feeding and respiration. The patient
failed extubation after the release of the mouth floor. Due to high risk
for aspiration and continued respiratory issues, gastrostomy tube
(G-tube) with fundoplication and tracheostomy were performed at 6 weeks
of life.

In addition to the feeding and respiratory issues, there was suspicion
of partial diabetes insipidus due to persistent hypernatremia and low
urine osmolarityand specific gravity. Brain MRI showed suspicion of
ectopic neurohypophysis but was otherwise normal. The patient also had
episodes of hypoglycemia requiring continuous feeds and treatment with
diazoxide. Insulin, growth hormone, free fatty acids, cortisol, and
\selectlanguage{greek}β-\selectlanguage{english}hydroxybutyrate random levels were unremarkable, although a critical
sample was unable to be collected during hypoglycemic events.

Initial genetic work-up included normal chromosome analysis, chromosome
microarray, spinal muscular atrophy analysis, and Fragile X testing. The
patient and both of his parents were then enrolled in the protocol for
rapid whole genome sequencing (rWGS).

\emph{Patient 2}

Patient 2 is a full-term AGA male born via repeat C-section at a local
hospital after an uncomplicated pregnancy. Upon delivery, he presented
as floppy and apneic with respiratory distress requiring intubation and
transfer to Nicklaus Children's Hospital. Prior to transfer, he was
placed on a nasal cannula after self-extubating.

Physical exam was significant for frontal bossing, bitemporal narrowing,
hypertelorism, deep set eyes, wide and depressed nasal bridge, right cup
ear, micrognathia, bilateral cryptorchidism, clenched hands with
overlapping fingers and toes, arthrogryposis multiplex congenita
(proximal and distal contractures of bilateral upper and lower
extremities),sacral dimple; peripheral hypertonia of upper and lower
extremities with central hypotonia, andabsent Moro reflex.

Evaluations during admission included normal chest x-ray, spine/hip/head
ultrasounds, brain MRI, CT skull, electroencephalogram (EEG) and
ophthalmologic evaluation. Abdominal ultrasound showed mild left
pelviectasis. Testicular ultrasound revealed left undescended testicle
and left inguinal hernia. Echocardiogram revealed a small atrial septal
defect. Early polysomnograms demonstrated central apnea.

Clinical course was complicated by gastroesophageal reflux disease
(GERD), respiratory distress requiring nasal cannula, apneic episodes
requiring caffeine administration, and poor feeding (NG tube
dependency). G-tube tube placement was performed at 6 weeks of age after
the videofluoroscopy swallow study showed silent aspiration of thin
liquids and significant pooling within the vallecula and piriform
sinuses.

Initial genetic work-up included normal newborn screen, chromosome
analysis, microarray and a nondiagnostic Neuromuscular Disorders Panel
at GeneDx Laboratory(80 genes). At 7 weeks of age, the patient and his
mother were enrolled for rapid whole genome sequencing (rWGS).

\textbf{METHODOLOGY}

\emph{Whole-Genome Sequencing} Whole genome sequencing was performed on
NovaSeq 6000 instruments (Illumina), generating paired 101-nt reads with
an average coverage of 55\selectlanguage{ngerman}× and 42x for proband 1 and 2 respectively.
Alignment and single nucleotide polymorphisms (SNV) calling were
performed using the DRAGEN processor (Illumina). Copy number variant
(CNV) calling was performed with CNVnator and Manta (Abyzov et al. 2011;
Chen et al. 2016). Variant call format (VCF) files incorporating SNV and
CNV calls were annotated and analyzed using Fabric Enterprise version
6.2.8 (Fabric Genomics) according to standard guidelines (Coonrod et al.
2013; Richards et al. 2015). The Human Phenotype Ontology terms used
during analysis are listed below. Parental samples for were available
for targeted variant analysis and inheritance determination.

Human Phenotype Ontology terms used for Patient 1: Neonatal hypotonia
(HP:0001319), Feeding difficulties in infancy (HP:0008872),
Arthrogryposis multiplex congenital (HP:0002804), Hip dysplasia
(HP:0001385), Overlapping fingers and toes (HP:0010557).

Human Phenotype Ontology terms used for Patient 1: Small for gestational
age (HP:0001518), Apnea (HP:0002104), Arthrogryposis multiplex
congenital (HP:0002804), Bilateral talipes equinovarus (HP:0001776) ,
Abnormality of limb bone morphology (HP:0002813).\textbf{RESULTS}

Patients 1 and 2 were each found to have a heterozygous, de novo,
pathogenic frameshift variant, c.1996dupC; p. Gln666ProfsTer47 in
the\emph{MAGEL2} gene (\emph{Table 1. rWGS Results of Patient 1 and 2}
). This is also referred to as the c.187dupC; p.Gln63ProfsTer47
(NM\_019066.4) in the literature based on the transcript used. This
variant is expected to create a frameshift in the protein coding
sequence, leading to a truncated protein. This variant has been
previously reported in individuals with Schaaf-Yang syndrome and has
been classified as pathogenic in ClinVar (Variation ID: 190122).

Patient 1 also had a heterozygous, maternally inherited, pathogenic
variant, c.3130\_3149delACCCCTGC; p. Thr1044LeufsTer63, in
the\emph{ABCC8} gene. This variant is expected to create a frameshift in
the protein sequence, leading to a truncated protein. This variant has
been classified as pathogenic in ClinVar and Human Gene Mutation
Database (HGMD). Pathogenic variation in the \emph{ABCC8} gene has been
associated with autosomal dominant/recessive familial hyperinsulinemic
hypoglycemia-1 (HHF1, MIM: \#256450). \textbf{LONG TERM CLINICAL
OUTCOMES AFTER DIAGNOSIS}

\emph{Patient 1}

Patient 1 was discharged from NICU at 12 weeks of age with a G-tube and
tracheostomy. Dioxide was discontinued prior to discharge with no
further episodes of hypoglycemia. The family moved back to his country
of origin but return periodically to continue care with various
specialties at Nicklaus Children's.

Patient 1 is currently 4 years old, has global developmental delay,
short stature, is non-ambulatory and continues to be G-tube and
tracheostomy dependent. He is currently undergoing endocrine evaluation
for concerns of micropenis and persistent cryptorchidism (pending
surgery). However, care has been complicated and delayed because of
barriers to accessing care.

The patient underwent bilateral Ponseti casting for his bilateral
clubfeet with residual equinus and cavus for which he later required
bilateral open Achilles lengthenings (2019). However, he was noted to
have mild recurrence of bilateral cavus and equinus deformities during
his last orthopedic evaluation, for which he is uses full time orthoses.
Furthermore, he has reduced range of motion of knees and hips secondary
to arthrogryposis and is unable to stand.

\emph{Patient 2}

Patient 2 was discharged from the NICU on nasal cannula to be used
during sleep. Soon after discharge, he was readmitted several times for
breath holding spells and unusual rigid movements but had normal repeat
EEG and negative work up.

Currently, he is 4 years of age, has global developmental delay, is
non-ambulatory, non-verbal and G-tube dependent. Since NICU discharge,
he is no longer having apneic episodes during sleep and oxygen was
discontinued. Ophthalmology has since noted esotropia, bilateral myopia
and astigmatism. He is also being followed for short stature, obesity,
GERD, and he has undergone strabismus surgery, tonsillectomy
adenoidectomy, and bilateral orchiopexy.

Patient 2 continues to have mild torticollis and mild knee flexion
contractures bilaterally managed with custom solid orthoses and physical
therapy. He also receives occupational and feeding therapies and has
nursing at home.

\textbf{DISCUSSION}

\emph{MAGEL2} is a paternally expressed, maternally imprinted,
protein-coding gene located on chromosome 15. \emph{MAGEL2} is one of
the genes that falls into the paternally expressed region of chromosome
15 (15q11-q13) known to result in Prader-Willi Syndrome (PWS, MIM:
\#176270)\textsuperscript{8}. Prader-Willi Syndrome is characterized by
hypotonia, feeding difficulties followed by hyperphagia later, failure
to thrive, developmental delay and, hypogonadism in
males.\textsuperscript{9} Schaaf-Yang Syndrome (SYS) has a great deal of
clinical overlap with PWS in that affected individuals often have
hypotonia, feeding difficulties, failure to thrive, developmental
delay/intellectual disabilities, and
hypogonadism.\textsuperscript{8}However, individuals with Schaaf Yang
are more likely to have congenital contractures ranging from mild to
severe, autistic spectrum disorder and
hypopituitarism.\textsuperscript{10,11,12} Hyperphagia which is common
in PWS is not a common feature of SYS. Truncating mutations
in\emph{MAGEL2} appear to be more consistent with classic SYS phenotype,
whereas whole gene deletions including the promoter region
in\emph{MAGEL2} have been found to have milder phenotypic
consequences.\textsuperscript{13,14} This may be due to a dominant
negative effect of the truncated protein or due to `leaky' expression of
the maternal copy of \emph{MAGEL2} in whole gene deletion cases which
has been suggested in mice studies \textsuperscript{9,12}.

\textbf{Comparing patient one and patient two}

Both our patients had many of the characteristic features reported in
SYS such as short stature, dysmorphic facial features, hypogonadism,
arthrogryposis multiplex congenital (AMC), hypotonia, respiratory
depression with apneic episodes, feeding difficulties requiring g-tube
placement, and delayed psychomotor
development\textsuperscript{3,16}(Table 2. Comparative table of clinical
features presented by Patient 1 and Patient 2). Nonetheless, there were
some notable differences between both patients. Patient 1 had IUGR and a
birth weight in the 5\textsuperscript{th} percentile while Patient 2 had
a birth weight in the 75\textsuperscript{th} percentile. Patient 1 is
tracheostomy dependent and had more severe congenital contractures while
patient 2 only required invasive respiratory support for a short period
and had milder contractures primarily involving the hands. Patient 1
also had unexplained hypernatremia and persistent hypoglycemia. However,
it is unknown whether these endocrine abnormalities were associated to
the MAGEL2 variant or whether the ABCC8 variant, found as an incidental
finding, may have contributed to these clinical features.

MacArthy et al. 2018, reported an association between the phenotypic
severity and the location of the truncating mutation and, described a
genotype-phenotype association comparing individuals with the
p.Gln666ProfsTer47 \emph{MAGEL2} variant with other mutations. According
to their prediction, this variant can be associated with severe
phenotypes, including a higher prevalence of joint contractures; more
severe feeding difficulties likely to require a nasogastric feeding
tube; more severe respiratory difficulties likely to require mechanical
ventilation and; more likely to have profound intellectual
disability/significant developmental delay.\textsuperscript{3} Both
patient 1 and patient 2 carry the p.Gln666ProfsTer47 variant
in\emph{MAGEL2} . Both had congenital contractures, required NG/G-tube
placement, invasive respiratory support, and had significant
developmental delays supporting the phenotype-genotype association
described by MacArthy et al. 2018.

It is important to note that hypopituitarism is reported as typically
present in patients with SYS but neither of the two patients have
presented with it. Likewise, hyperphagia has been reported to be present
in 22\% of reported cases\textsuperscript{3} and, although patient 2 has
been noted to be overweight, it is unclear if hyperphagia is present or
not.

\textbf{Current needs in the SYS research world}

The current knowledge available about the natural presentation and
course of SYS is limited. In January 2019, the SYS/MAGEL2 Advisory Group
outlined the needs of SYS research, which include identifying the full
phenotypic spectrum of SYS, establishing standards of care for
individuals with SYS, and understanding the SYS patient's perspective to
improve outcomes \textsuperscript{17}.

While patient 1 and patient 2 presented close in time with similar
clinical presentation, enough differences existed to keep the patients
distinct. It was the utilization of rapid whole genome sequencing that
found the commonality between these two patients, which might have not
been achieved based on clinical criteria only. McCarthy et al, 2018
noted that based on clinical presentation, several different conditions
may be caused by truncating mutations in \emph{MAGEL2} . In fact,
individuals initially diagnosed with Opitz Trigonocephaly C syndrome,
Chitayat-Hall syndrome, and Arthrogryposis Multiplex Complex diagnoses
were found to have truncating mutations in
MAGEL2.\textsuperscript{18-21} Thus, many more cases of SYS may exist
that can add to the variable phenotypic picture that exists today.
Nevertheless, this wide phenotypic spectrum that is characteristic of
SYS represents an obstacle for reaching a diagnosis as patients
typically undergo extensive workup prior to achieving the diagnosis of
SYS. This is especially important considering that the \emph{MAGEL2}gene
is not included in many gene panels and is, therefore, typically only
identified through broad testing options (i.e. expanded panels, whole
exome sequencing (WES ) and WGS). For both of our patients, early
diagnosis was achieved through the rWGS protocol, which allowed for
rapid turnaround of results in the acute setting.

With whole genome sequencing becoming more
accessible\textsuperscript{22}, this diagnostic odyssey can be
shortened, saving both time and other valuable resources for families
and the community. An earlier diagnosis can also have a significant
impact in care outcomes by guiding decision-making in the acute care
setting, especially now with some available knowledge about certain
phenotype-genotype associations that could influence those decisions. An
example of this would be earlier tracheostomy placement based on the
expected prognosis associated with the p.Gln666ProfsTer47
\emph{MAGEL2}variant, as multiple studies (in both children and adults)
have reported improved medical outcomes associated with earlier
tracheostomy in critically ill patients.\textsuperscript{23-25}

\textbf{Conclusions}

Our patients exemplify the broad phenotypic spectrum that is
characteristic of SYS while at the same time support the previously
described phenotype-genotype association suggested by MacArthy et al.
2018, in which they associate the p.Gln666ProfsTer47
\emph{MAGEL2}variant with more severe clinical outcomes. e This report
supports the early utilization of whole genome sequencing, especially in
critical settings, to facilitate early diagnosis of rare conditions like
SYS, which, in turn, could help save time and resources and help to
improve clinical outcomes of patients.

\textbf{Abbreviations and Acronyms}

Arthrogryposis multiplex congenital (AMC)

Copy number variant (CNV)

Gastroesophageal reflux disease (GERD)

Gastrostomy tube (G-tube)

Intrauterine growth restriction (IUGR)

Nasogastric tube (NG tube)

Next Generation Sequencing (NGS)

Neonatal intensive care unit (NICU)

Prader-Willi Syndrome (PWS)

Rapid Whole Genome Sequencing (rWGS)

Schaaf-Yang Syndrome (SYS)

Single nucleotide polymorphisms (SNV)

Small for gestational age (SGA)

Whole Genome Sequencing (WGS)

\textbf{Acknowledgements}

Would like to thank Nicklaus Children's and RCIGM team Michelin Janvier,
Leonardo Villaroel, Bala Totapally, Jun Sasak. RCIGM team for sequencing
support.

\textbf{Author Contributions:}

Ana Maria Rodriguez: Conceptualization; Investigation; Writing -
Original Draft; Visualization

Katherine Schain: Conceptualization; Investigation; Writing - Original
Draft

Parul Jayakar: Conceptualization; Writing - Original Draft;
Visualization; Supervision; Project administration Meredith S. Wright:
Formal analysis; Investigation; Resources; Writing - Original Draft

Shimul Chowdhury: Formal analysis; Investigation; Resources; Writing -
Original Draft

Daria Salyakina: Writing - Original Draft; Visualization; Supervision;
Project administration.

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\end{enumerate}

\textbf{Hosted file}

\verb`Tables. Schaaf-Yang Syndrome.docx`  available at \url{https://authorea.com/users/622938/articles/645849-report-of-two-cases-of-schaaf-yang-syndrome-same-genotype-different-phenotype}

\textbf{Hosted file}

\verb`Table 2 SYS.docx`  available at \url{https://authorea.com/users/622938/articles/645849-report-of-two-cases-of-schaaf-yang-syndrome-same-genotype-different-phenotype}

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