Truncated 
Sperm-associated antigen 9 ( SPAG9 ) was cloned in 1998 and called a
protein highly expressed in testis (PHET) ( ). SPAG9 has been implicated in signal transduction in
interacting with the JNK (c-Jun N-terminal kinase) signaling pathway, which is
involved in cell survival, apoptosis, stress responses and plays a vital role in
maintenance of cell shape, motility, and intracellular transport. SPAG9 is known to be expressed in various tissues, including
the testes, which is consistent with its original identification as a
sperm-associated antigen. It’s worth noting that SPAG9’s expression
might vary in different cell types and tissues ( ). SPAG9 is a JNK-associated leucine zipper protein (JLP) and with JNK/stress-activated
protein kinase-associated protein 1 (JSAP1 or MAPK8IP3), which are structurally
related scaffolding proteins highly expressed in the brain. JIP4 is a scaffold
protein that in humans is encoded by the SPAG9 gene. SPAG9 plays
functionally redundant and essential roles in mouse cerebellar Purkinje cell (PC)
survival ( ). Mice
containing PCs with deletions in both JSAP1 and JLP exhibited PC axonal dystrophy,
followed by gradual, progressive neuronal loss. These findings suggest that JSAP1
(MAPK8IP3) and JLP play critical roles in kinesin-1-dependent axonal transport,
which prevents brain neuronal degeneration ( , ). With structural homology of JNK, SPAG9 is
involved in MAPK signaling pathway to regulate cellular activities ( ). Oculocutaneous albinism (OCA) is a heterogeneous group disorder that results in
either reduction or complete loss of melanin formation of melanin in melanocytes,
which results in mild to severe hypopigmentation of the hair, skin, and eyes. OCA is
classified into two subtypes, non-syndromic OCA and syndromic OCA. Non-syndromic OCA
is caused by disease causing variants in genes associated with melanocyte
differentiation, melanosomal proteins, and melanin synthesis, which cause only
hypopigmentation and visual-associated symptoms ( ). Currently, there are eight types of
non-syndromic OCA that have been reported: OCA1 to OCA8 and the most common forms
are TYR-related types and include OCA type 1A (MIM 203100) and OCA type 1B (MIM
606952) ( ; ; ; ; ; ).
Furthermore, syndromic OCA includes systemic phenotypic manifestations such as
intellectual disability, global developmental delay, and cataract. Syndromic OCA is
caused by mutations in cargo trafficking proteins, which contribute to the formation
of lysosome-related organelles (LROs). LROs are cell type-specific organelles, such
as melanosomes in melanocytes, which cause the classic OCA phenotype of skin, eye,
and hair hypopigmentation. Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi
syndrome (CHS) are the two most common types of syndromic OCA ( ). Only cutaneous and ocular signs and symptoms in OCA patients have been studied; its
association with brain development and other associated phenotypes has not been
studied well. As we know that disruption of the tyrosine pathways involved in the OCA are essential
for the retinal and visual network development, however indirectly, that are also
linked to progressive neurodevelopment in the patients ( ). In the present study, we aim to describe a novel syndrome for the first time in the
literature, and we link an SPAG9 gene defect as a possible cause of
this syndrome (CACD syndrome). We performed genetic, clinical and molecular
characterization of two families to find the associated genes and further performed
functional study (RT-qPCR) and 3D protein modeling to examine the pathogenicity of
the identified variant. 

Ethics approval and consent to participate The study was approved by the Institutional Review Board (IRB) of King Abdullah
International Medical Research Centre (KAIMRC), Riyadh [Grant # RC19/138/R;
IRBC/1580/20]. The parents of the patient gave written informed consent for
publications of data and images in accordance with the Declaration of Helsinki.
Written informed consent for the publication of related data was obtained from
the parents of the enrolled patients. Patient recruitment and DNA extraction Two families A and B from Saudi Arabian population with a severe autosomal
recessive oculocutaneous albinism were recruited for the current investigation
( , ). Both families were unrelated according to the family
history. A detailed medical history was obtained, magnetic resonance imaging
(MRI), and biochemical tests were performed ( - ). Blood samples
were obtained from both families ( , ) and processed further for DNA
extraction and quantification using conventional techniques ( ). Molecular Investigation Whole genome sequencing (WGS) was performed using standard methods on DNA from
both families. The Twist Human Core Exome Plus kit was used to enrich target
regions from fragmented genomic DNA by using double-stranded DNA capture baits
against approximately 36.5 Mb of the human coding exome (targeting >98% of
the coding RefSeq: GRCh37/hg19). The generated library was sequenced on an
Illumina platform to achieve at least 20× coverage depth for over 98%. We used
an in-house bioinformatics pipeline that included read alignment to the
GRCh37/hg19, variant-calling, annotation, and comprehensive variant filtering.
The variant calling file (VCF) files were analyzed using Illumina BaseSpace, and
different filters were applied based on clinical information and family history
( ; ). All variants with a minor allele frequency (MAF) of less than 1% in the gnomAD
database, as well as disease-causing variants from HGMD®, ClinVar, or CentoMD®,
were considered. The search for relevant variants was restricted to coding exons
and flanking +/-20 intronic nucleotides of genes with clear gene-phenotype
evidence (OMIM®). All possible modes of inheritance patterns were considered;
however, based on the pedigree, autosomal recessive was prioritized. ACMG
classifies variants into five categories (pathogenic, likely pathogenic, variant
of uncertain significant (VUS), likely benign, and benign) ( ). Using standard
screening procedures, we looked for functional variations that could be
associated with the patient phenotype. Priority was given to genes that have
previously been described in the literature (PUBMED). In silico analysis Several techniques were used to determine the discovered variant’s pathogenic
potential. To determine if the variant is reported in the general population or
not, gnomAD were searched. Using NCBI-HomoloGene, amino acid conservation was
determined. Sanger sequencing The observed variant was Sanger sequenced in all accessible members of the
family. Sanger sequencing was carried out using standard procedures ( ).
Primer pairs were created using an online software application called Primer3.
Primer sequence for the Sanger sequencing:
[ SPAG9 -903-F1-TTAGCCAAGGCGGATCTAAA, SPAG9 -903-R1-TCCTGGGCTACCTGTACTTCA] ( F). RNA extraction PBMCs were used to isolate total RNA, which was then separated into its organic
and aqueous phases using the TRIzol® reagent and chloroform. The RNA from the
aqueous phase was transferred to the RNase-free tube after spinning at 4 °C for
15 minutes. After washing with isopropanol, precipitation was done using 75%
ethanol. Total RNA was extracted using the RNeasy Plus Mini Kit from Qiagen
Inc., purity tests for RNA and quantification were done using conventional
methods ( ; ). Quantitative real-time PCR [qRT-PCR] Total RNA was collected to quantify the expression of SPAG9 mRNA
in comparison to the internal control gene ( GAPDH ). The
high-capacity cDNA reverse transcription kit (Applied Biosystems) was used to
create cDNA using standard techniques from total RNA ( ; ). The SPAG9 cDNA primer sequences were created using the
Primerbank database [ SPAG9 -cDNA-F1: TCTGATGTTAGCCAAGGC, SPAG9 -cDNA-R1: TTCCTGGGCTACCTGTAC]. Thermo Fisher’s PCR
SYBRGreen Master Mix was used in the qPCR reaction, which was run on an Applied
Biosystems QuantStudio 6 Flex Real-Time PCR System. The expression Suite
software, version 1.1 (Applied Biosystems), was used to analyze the data after
each reaction was independently replicated and carried out in triplicate. GAPDH was used as the endogenous control, and the PCR cycle
settings were according to standard protocols. GraphPad Prism (version 8.1) was
used to analyze the quantitative real-time PCR results as the mean standard
deviation (SD). For significance evaluation, a t-test with a threshold of p <
0.01 was employed on the samples. SPAG9 sequence retrieval and 3D structure prediction In this study, in silico methodologies such as homology modeling
for wild-type and mutant were carried out. The crystal structure of the human
C-Jun-amino-terminal kinase-interacting protein 4 (JIP4) was retrieved from the
AlphaFold Protein Structure Database (AlphaFold DB), and the Swiss-Model was
used to create the structure of the mutated protein. The protein structure from
the AlphaFold DB was used as a template to yield the mutated protein structure.
The model was then run through the Ramachandran plot server and ERRAT. STRING
was used to predict protein-protein interactions
(https://www.expasy.org/resources/string). Electron microscopic (EM) Electron microscopic (EM) studies were performed including high resolution
imaging of ultra-structural organelles using standard protocols. The specimens
were received in glutaraldehyde (to preserve cellular structures at the
ultrastructural level). After fixation, the samples are dehydrated with
increasing concentrations of alcohol and embedded in resin for sectioning. It
was labeled “skin punch biopsy” and consists of a single tiny piece of grayish
white soft tissue measuring 0.2 × 0.1 cm in diameter and 0.2 cm in depth.
Samples were stained by using uranyl acetate and lead citrate and examined under
electron microscope. 

The study was approved by the Institutional Review Board (IRB) of King Abdullah
International Medical Research Centre (KAIMRC), Riyadh [Grant # RC19/138/R;
IRBC/1580/20]. The parents of the patient gave written informed consent for
publications of data and images in accordance with the Declaration of Helsinki.
Written informed consent for the publication of related data was obtained from
the parents of the enrolled patients. 

Two families A and B from Saudi Arabian population with a severe autosomal
recessive oculocutaneous albinism were recruited for the current investigation
( , ). Both families were unrelated according to the family
history. A detailed medical history was obtained, magnetic resonance imaging
(MRI), and biochemical tests were performed ( - ). Blood samples
were obtained from both families ( , ) and processed further for DNA
extraction and quantification using conventional techniques ( ). 

Whole genome sequencing (WGS) was performed using standard methods on DNA from
both families. The Twist Human Core Exome Plus kit was used to enrich target
regions from fragmented genomic DNA by using double-stranded DNA capture baits
against approximately 36.5 Mb of the human coding exome (targeting >98% of
the coding RefSeq: GRCh37/hg19). The generated library was sequenced on an
Illumina platform to achieve at least 20× coverage depth for over 98%. We used
an in-house bioinformatics pipeline that included read alignment to the
GRCh37/hg19, variant-calling, annotation, and comprehensive variant filtering.
The variant calling file (VCF) files were analyzed using Illumina BaseSpace, and
different filters were applied based on clinical information and family history
( ; ). All variants with a minor allele frequency (MAF) of less than 1% in the gnomAD
database, as well as disease-causing variants from HGMD®, ClinVar, or CentoMD®,
were considered. The search for relevant variants was restricted to coding exons
and flanking +/-20 intronic nucleotides of genes with clear gene-phenotype
evidence (OMIM®). All possible modes of inheritance patterns were considered;
however, based on the pedigree, autosomal recessive was prioritized. ACMG
classifies variants into five categories (pathogenic, likely pathogenic, variant
of uncertain significant (VUS), likely benign, and benign) ( ). Using standard
screening procedures, we looked for functional variations that could be
associated with the patient phenotype. Priority was given to genes that have
previously been described in the literature (PUBMED). 

analysis Several techniques were used to determine the discovered variant’s pathogenic
potential. To determine if the variant is reported in the general population or
not, gnomAD were searched. Using NCBI-HomoloGene, amino acid conservation was
determined. 

The observed variant was Sanger sequenced in all accessible members of the
family. Sanger sequencing was carried out using standard procedures ( ).
Primer pairs were created using an online software application called Primer3.
Primer sequence for the Sanger sequencing:
[ SPAG9 -903-F1-TTAGCCAAGGCGGATCTAAA, SPAG9 -903-R1-TCCTGGGCTACCTGTACTTCA] ( F). 

PBMCs were used to isolate total RNA, which was then separated into its organic
and aqueous phases using the TRIzol® reagent and chloroform. The RNA from the
aqueous phase was transferred to the RNase-free tube after spinning at 4 °C for
15 minutes. After washing with isopropanol, precipitation was done using 75%
ethanol. Total RNA was extracted using the RNeasy Plus Mini Kit from Qiagen
Inc., purity tests for RNA and quantification were done using conventional
methods ( ; ). 

Total RNA was collected to quantify the expression of SPAG9 mRNA
in comparison to the internal control gene ( GAPDH ). The
high-capacity cDNA reverse transcription kit (Applied Biosystems) was used to
create cDNA using standard techniques from total RNA ( ; ). The SPAG9 cDNA primer sequences were created using the
Primerbank database [ SPAG9 -cDNA-F1: TCTGATGTTAGCCAAGGC, SPAG9 -cDNA-R1: TTCCTGGGCTACCTGTAC]. Thermo Fisher’s PCR
SYBRGreen Master Mix was used in the qPCR reaction, which was run on an Applied
Biosystems QuantStudio 6 Flex Real-Time PCR System. The expression Suite
software, version 1.1 (Applied Biosystems), was used to analyze the data after
each reaction was independently replicated and carried out in triplicate. GAPDH was used as the endogenous control, and the PCR cycle
settings were according to standard protocols. GraphPad Prism (version 8.1) was
used to analyze the quantitative real-time PCR results as the mean standard
deviation (SD). For significance evaluation, a t-test with a threshold of p <
0.01 was employed on the samples. 

sequence retrieval and 3D structure prediction In this study, in silico methodologies such as homology modeling
for wild-type and mutant were carried out. The crystal structure of the human
C-Jun-amino-terminal kinase-interacting protein 4 (JIP4) was retrieved from the
AlphaFold Protein Structure Database (AlphaFold DB), and the Swiss-Model was
used to create the structure of the mutated protein. The protein structure from
the AlphaFold DB was used as a template to yield the mutated protein structure.
The model was then run through the Ramachandran plot server and ERRAT. STRING
was used to predict protein-protein interactions
(https://www.expasy.org/resources/string). 

Electron microscopic (EM) studies were performed including high resolution
imaging of ultra-structural organelles using standard protocols. The specimens
were received in glutaraldehyde (to preserve cellular structures at the
ultrastructural level). After fixation, the samples are dehydrated with
increasing concentrations of alcohol and embedded in resin for sectioning. It
was labeled “skin punch biopsy” and consists of a single tiny piece of grayish
white soft tissue measuring 0.2 × 0.1 cm in diameter and 0.2 cm in depth.
Samples were stained by using uranyl acetate and lead citrate and examined under
electron microscope. 

Clinical Description Family A The affected individual in family A (II-2) was a 4 years old female, known to
have G6PD deficiency, second child of consanguineous parents ( C). The patient was born following
uneventful gestation and delivery, at term. Her APGAR score was 9 and 9 at 1 and
5 minutes respectively, birth weight was 3.4 kg (25 th -50 th percentile), length was 53 cm (90 th percentile) and head
circumference 34.2 cm (25 th -50 th percentile), and found to
have oculocutaneous albinism since birth and she was discharged with her mother
in good health. At age of 6 months, she presented with cataract and
developmental delay, mainly motor as she has poor head control and cannot sit
without support. At one year of age, she underwent cataract surgery and
developmentally still cannot sit or crawl and has no speech. She had normal
hearing. Her past medical history was consistent of two attacks of pneumonia,
one of which was at age 20 days with neonatal intensive care unit (NICU)
admission and the other admission was at the age of 2 years. As for her current
development (age 4 years), she still cannot walk, she can only sit and crawl.
Her soft motor skills are delayed as well; she cannot draw shapes, feed herself,
nor help in dressing or undressing or control her sphincters. As for her speech,
she can only call her parents, but no other understandable words. Moreover, she
cannot follow any commands and does not recognize her siblings well. She is
developmentally functioning at age of 9 months. At her last physical evaluation; the affected individual had weight of 13 kg
(3 rd -5th percentile), length was 91 cm (<3percentile) and head
circumference 49 cm (25 th -50 th percentile). General
examination revealed features of coarse facial dysmorphism (Ocular hypertelorism
with prominent supraorbital rim, low-set ears, broad nose with flaring of
nostrils, deeply grooved philtrum with macroglosia), Eye examination showed
normal iris pigmentation, full extra-ocular movement, exotropia at distance in
left eye and bilateral positive red reflex, no nystagmus., generalized skin
hypopigmentation and blond-gray hair, eyelashes and eyebrow. She was also noted
to have generalized hypotonia with normal power and reflexes ( ). Other system examination were
unremarkable. Her echocardiogram showed tiny foramen ovale and mild tricuspid
valve regurgitation. Abdomen ultrasound revealed diffuse increased parenchymal
liver echogenicity. The skeletal survey was remarkable for mild decrease in bone
density and bilateral developmental dysplasia of the hip (DDH) with bilateral
coxa valga deformity. Brain Magnetic Resonance Imaging (MRI) was notable for
diffuse bilateral supratentorial pachygyria, predominantly involving the frontal
lobes ( ). Family B The affected individual in family B (II-2) is 8 years old female born to a
consanguineous parent ( ). She is
a product of full term, delivered by caesarian section (C\S) due to failure to
progress. The fetus (II-1) had a spontaneous abortion; however, the reason was
not documented. The patient was born following uneventful gestation and
delivery, at term. Her APGAR score was 9 and 9 at 1 and 5 minutes respectively,
birth weight was 3 kg (25 th -50 th percentile), length was
52 cm (75 th -90th percentile) and head circumference 34 cm
(25 th -50 th percentile), Since birth she was noted to
have oculocutaneous albinism, and was admitted to nursery to rule out sepsis
with respiratory difficulties given oxygen through nasal cannula and discharged
after 10 days with diagnosis of transient tachypnea of newborn. Since discharge,
she had hypotonia, feeding difficulties, and frequent vomiting. Also, at 6
months of age, the family noted decreased visual acuity, and her
ophthalmological assessment revealed that she had cataract, which was operated
on at the age of 10 months. In addition, she has had global developmental delay
mainly motor as she has poor head control and cannot sit without support. She
sat at two years of age, crawled at 2 year 2 months of age and walked
independently at 4 years of age. She also had delayed speech and her hearing was
normal. Extensive investigations at the time did not reveal a genetic cause for
her presentation including: chromosomal analysis, CGH microarray and whole exome
sequencing. Currently she is having global developmental delay, she can ambulate
independently on flat surfaces; however, she still needs support to climb up or
down the stairs. As for her fine motor (age 8 years), she can only scribble with
a pen and cannot draw shapes or letters. She cannot feed herself nor she can
maintain her personal hygiene and she is not toilet trained. She needs help in
all daily life activities. She can now speak two-words sentences with around 50%
understandable words; she can follow only few simple commands. She
developmentally functioning at 3 years of age. She also has recurrent otitis
media and ear effusion with sleep apnoea; therefore, she underwent adenoidectomy
with bilateral myringotomy and right ventilation tube (VT) insertion at 6 years
of age. At her last physical evaluation; she had weight of 30 kg
(50 th -75 th percentile), height was 128 cm
(25 th -50 th percentile) and head circumference 51 cm
(25 th -50 th percentile). She was noted to have features
of coarse facial dysmorphism (Ocular hypertelorism, downward slant of palpebral
fissure, broad nose with depressed root, deeply grooved philtrum with full
lips). Eye examination showed normal iris pigmentation, she is wearing glasses
with full extra-ocular movement, squint and bilateral positive red reflex and
nystagmus. She has generalized skin hypopigmentation and blond-yellow hair,
eyelashes and eyebrow. She was also noted to have generalized hypotonia with
normal power and reflexes. The skeletal survey showed right sided developmental
dysplasia of the hip (DDH). Hearing assessment was done and was remarkable for
bilateral abnormal middle ear function. Her echocardiogram was positive for
small patent ductus arteriosus (PDA) and mild tricuspid as well as mitral valves
regurgitation. Abdomen ultrasound revealed heterogeneous liver echotexture.
Brain MRI and nerve conduction studies were unremarkable. Electron microscopy of
the index skin biopsy suggesting melanocyte with melanosomes of variable stages
of development some are lacking melanin pigmentation. Electron microscopy of the
index skin biopsy suggesting melanocyte with melanosomes of variable stages of
development some are lacking melanin pigmentation (pale color; , ). Biochemical investigations All biochemical investigations were unremarkable including: acylcarnitine
profile, plasma amino acids, urine organic acids, creatine kinase (CK) level,
total homocysteine, lactic acid, ammonia level, carbohydrate deficient
transferrin (CDT) for both the families (A, B). Molecular investigation The WGS and variants filtration stages were carried for both the families using
the previously published standard procedures ( ). A novel homozygous frameshift variant
[c.903del; p.(Phe301Leufs*2)] was identified in both the families in the exon 6
of the SPAG9 gene located on chromosome 17q21.33 [NM_003971.6].
The variant [c.903del; p.(Phe301Leufs*2)] was discovered after screening and
filtering several homozygous and compound heterozygous variants. The SPAG9 variant [c.903del; p.(Phe301Leufs*2)] creates a shift
in the reading frame starting at codon 301 ( , ). The new reading frame
ends in a stop codon 1 positions downstream. Sanger sequencing was performed for
both families A and B and the variant (c.903del) segregated perfectly with the
disease phenotype ( ). Moreover,
1500 control genomes were screened for the mutation, and it was discovered that
the altered amino acid was conserved in all tested genomes. According to ACMG
categorization, the discovered variation is categorized as a variant of
uncertain significance (VUS) class 3. Quantitative qPCR The SPAG9 mRNA expression was examined using RT-qPCR in both
families A and B including the probands, both parents, and normal individuals.
The RT-qPCR data was analyzed and revealed that the probands (Family A (II-2)
and Family B (II-2) having the homozygous variant (c.903del) revealed a
significant reduction in the relative mRNA expression of the SPAG9 as compared to the heterozygous parents and wildtype
controls ( C, D). SPAG9 -3D protein modeling The JIP4 protein has a transmembrane binding site, coiled coil regions, a leucine
zipper motif, and a JNK binding motif. The Phenylalanine (F) to Leucine (L)
codon substitution brought about by the frameshift mutation that finally led to
the protein chain’s termination at amino acid number 302. As a result, it is
anticipated that the modified structure will stop at 302 ( ). Numerous interactions between these domains and
proteins are known to occur. The final revised model was examined by several
evaluation programs. According to the Ramachandran plot, the allowed torsion
angle zones are occupied by 97% and 99% of the residues in the wild-type and
mutant structures, respectively ( ). It is plausible that SPAG9 isoforms might interact with proteins that regulate
the trafficking of melanosomes or melanosomal proteins required for appropriate
pigmentation since they can interact with proteins like ARF6 , MAPK8 , MAPK8IP1 , SPAM1 , HYAL3 , HYAL1 , SMCO1 and PIP4P1 that are involved in certain types of vesicular
trafficking ( ). Melanins, the
colors for skin, hair, and eyes, are created in melanosomes and then carried to
the terminals of melanocyte dendrites and exported to nearby keratinocytes
through microtubule and actin filaments. The shortened protein caused by the
mutation (p.Phe301Leufs*2) is unable to interact with other proteins. 

Family A The affected individual in family A (II-2) was a 4 years old female, known to
have G6PD deficiency, second child of consanguineous parents ( C). The patient was born following
uneventful gestation and delivery, at term. Her APGAR score was 9 and 9 at 1 and
5 minutes respectively, birth weight was 3.4 kg (25 th -50 th percentile), length was 53 cm (90 th percentile) and head
circumference 34.2 cm (25 th -50 th percentile), and found to
have oculocutaneous albinism since birth and she was discharged with her mother
in good health. At age of 6 months, she presented with cataract and
developmental delay, mainly motor as she has poor head control and cannot sit
without support. At one year of age, she underwent cataract surgery and
developmentally still cannot sit or crawl and has no speech. She had normal
hearing. Her past medical history was consistent of two attacks of pneumonia,
one of which was at age 20 days with neonatal intensive care unit (NICU)
admission and the other admission was at the age of 2 years. As for her current
development (age 4 years), she still cannot walk, she can only sit and crawl.
Her soft motor skills are delayed as well; she cannot draw shapes, feed herself,
nor help in dressing or undressing or control her sphincters. As for her speech,
she can only call her parents, but no other understandable words. Moreover, she
cannot follow any commands and does not recognize her siblings well. She is
developmentally functioning at age of 9 months. At her last physical evaluation; the affected individual had weight of 13 kg
(3 rd -5th percentile), length was 91 cm (<3percentile) and head
circumference 49 cm (25 th -50 th percentile). General
examination revealed features of coarse facial dysmorphism (Ocular hypertelorism
with prominent supraorbital rim, low-set ears, broad nose with flaring of
nostrils, deeply grooved philtrum with macroglosia), Eye examination showed
normal iris pigmentation, full extra-ocular movement, exotropia at distance in
left eye and bilateral positive red reflex, no nystagmus., generalized skin
hypopigmentation and blond-gray hair, eyelashes and eyebrow. She was also noted
to have generalized hypotonia with normal power and reflexes ( ). Other system examination were
unremarkable. Her echocardiogram showed tiny foramen ovale and mild tricuspid
valve regurgitation. Abdomen ultrasound revealed diffuse increased parenchymal
liver echogenicity. The skeletal survey was remarkable for mild decrease in bone
density and bilateral developmental dysplasia of the hip (DDH) with bilateral
coxa valga deformity. Brain Magnetic Resonance Imaging (MRI) was notable for
diffuse bilateral supratentorial pachygyria, predominantly involving the frontal
lobes ( ). Family B The affected individual in family B (II-2) is 8 years old female born to a
consanguineous parent ( ). She is
a product of full term, delivered by caesarian section (C\S) due to failure to
progress. The fetus (II-1) had a spontaneous abortion; however, the reason was
not documented. The patient was born following uneventful gestation and
delivery, at term. Her APGAR score was 9 and 9 at 1 and 5 minutes respectively,
birth weight was 3 kg (25 th -50 th percentile), length was
52 cm (75 th -90th percentile) and head circumference 34 cm
(25 th -50 th percentile), Since birth she was noted to
have oculocutaneous albinism, and was admitted to nursery to rule out sepsis
with respiratory difficulties given oxygen through nasal cannula and discharged
after 10 days with diagnosis of transient tachypnea of newborn. Since discharge,
she had hypotonia, feeding difficulties, and frequent vomiting. Also, at 6
months of age, the family noted decreased visual acuity, and her
ophthalmological assessment revealed that she had cataract, which was operated
on at the age of 10 months. In addition, she has had global developmental delay
mainly motor as she has poor head control and cannot sit without support. She
sat at two years of age, crawled at 2 year 2 months of age and walked
independently at 4 years of age. She also had delayed speech and her hearing was
normal. Extensive investigations at the time did not reveal a genetic cause for
her presentation including: chromosomal analysis, CGH microarray and whole exome
sequencing. Currently she is having global developmental delay, she can ambulate
independently on flat surfaces; however, she still needs support to climb up or
down the stairs. As for her fine motor (age 8 years), she can only scribble with
a pen and cannot draw shapes or letters. She cannot feed herself nor she can
maintain her personal hygiene and she is not toilet trained. She needs help in
all daily life activities. She can now speak two-words sentences with around 50%
understandable words; she can follow only few simple commands. She
developmentally functioning at 3 years of age. She also has recurrent otitis
media and ear effusion with sleep apnoea; therefore, she underwent adenoidectomy
with bilateral myringotomy and right ventilation tube (VT) insertion at 6 years
of age. At her last physical evaluation; she had weight of 30 kg
(50 th -75 th percentile), height was 128 cm
(25 th -50 th percentile) and head circumference 51 cm
(25 th -50 th percentile). She was noted to have features
of coarse facial dysmorphism (Ocular hypertelorism, downward slant of palpebral
fissure, broad nose with depressed root, deeply grooved philtrum with full
lips). Eye examination showed normal iris pigmentation, she is wearing glasses
with full extra-ocular movement, squint and bilateral positive red reflex and
nystagmus. She has generalized skin hypopigmentation and blond-yellow hair,
eyelashes and eyebrow. She was also noted to have generalized hypotonia with
normal power and reflexes. The skeletal survey showed right sided developmental
dysplasia of the hip (DDH). Hearing assessment was done and was remarkable for
bilateral abnormal middle ear function. Her echocardiogram was positive for
small patent ductus arteriosus (PDA) and mild tricuspid as well as mitral valves
regurgitation. Abdomen ultrasound revealed heterogeneous liver echotexture.
Brain MRI and nerve conduction studies were unremarkable. Electron microscopy of
the index skin biopsy suggesting melanocyte with melanosomes of variable stages
of development some are lacking melanin pigmentation. Electron microscopy of the
index skin biopsy suggesting melanocyte with melanosomes of variable stages of
development some are lacking melanin pigmentation (pale color; , ). 

All biochemical investigations were unremarkable including: acylcarnitine
profile, plasma amino acids, urine organic acids, creatine kinase (CK) level,
total homocysteine, lactic acid, ammonia level, carbohydrate deficient
transferrin (CDT) for both the families (A, B). 

The WGS and variants filtration stages were carried for both the families using
the previously published standard procedures ( ). A novel homozygous frameshift variant
[c.903del; p.(Phe301Leufs*2)] was identified in both the families in the exon 6
of the SPAG9 gene located on chromosome 17q21.33 [NM_003971.6].
The variant [c.903del; p.(Phe301Leufs*2)] was discovered after screening and
filtering several homozygous and compound heterozygous variants. The SPAG9 variant [c.903del; p.(Phe301Leufs*2)] creates a shift
in the reading frame starting at codon 301 ( , ). The new reading frame
ends in a stop codon 1 positions downstream. Sanger sequencing was performed for
both families A and B and the variant (c.903del) segregated perfectly with the
disease phenotype ( ). Moreover,
1500 control genomes were screened for the mutation, and it was discovered that
the altered amino acid was conserved in all tested genomes. According to ACMG
categorization, the discovered variation is categorized as a variant of
uncertain significance (VUS) class 3. 

The SPAG9 mRNA expression was examined using RT-qPCR in both
families A and B including the probands, both parents, and normal individuals.
The RT-qPCR data was analyzed and revealed that the probands (Family A (II-2)
and Family B (II-2) having the homozygous variant (c.903del) revealed a
significant reduction in the relative mRNA expression of the SPAG9 as compared to the heterozygous parents and wildtype
controls ( C, D). 

-3D protein modeling The JIP4 protein has a transmembrane binding site, coiled coil regions, a leucine
zipper motif, and a JNK binding motif. The Phenylalanine (F) to Leucine (L)
codon substitution brought about by the frameshift mutation that finally led to
the protein chain’s termination at amino acid number 302. As a result, it is
anticipated that the modified structure will stop at 302 ( ). Numerous interactions between these domains and
proteins are known to occur. The final revised model was examined by several
evaluation programs. According to the Ramachandran plot, the allowed torsion
angle zones are occupied by 97% and 99% of the residues in the wild-type and
mutant structures, respectively ( ). It is plausible that SPAG9 isoforms might interact with proteins that regulate
the trafficking of melanosomes or melanosomal proteins required for appropriate
pigmentation since they can interact with proteins like ARF6 , MAPK8 , MAPK8IP1 , SPAM1 , HYAL3 , HYAL1 , SMCO1 and PIP4P1 that are involved in certain types of vesicular
trafficking ( ). Melanins, the
colors for skin, hair, and eyes, are created in melanosomes and then carried to
the terminals of melanocyte dendrites and exported to nearby keratinocytes
through microtubule and actin filaments. The shortened protein caused by the
mutation (p.Phe301Leufs*2) is unable to interact with other proteins. 

In the present study, we genetically and clinically examined two unrelated families
each having single affected individual with oculocutaneous albinism features having
fair hair, fair skin, ocular abnormalities, cataract, developmental delay,
developmental dysplasia of the hips and coarse facial dysmorphism raising the
possibility of a new syndrome. To investigate the molecular cause, the affected
individuals were subjected to WGS using standard methods. WGS coupled with Sanger
sequencing revealed a novel frameshift variant (c.903del; p.Phe301Leufs*2) in exon 6
of the SPAG9 gene located on chromosome 17q21.33. SPAG9 has 29 coding exons, which encode a 1308 amino acid long
protein. Several disorders have been linked to ocular albinism, intellectual disability, and
other clinical manifestations. Hermansky-Pudlak syndrome (HPS; OMIM 608233),
Chediak-Higashi syndrome (CHS; OMIM 214500), and Nettleship-Falls syndrome
(Nettleship syndrome; OMIM 300500) are some common examples. We tested all of the
genes associated with these syndromes through WGS and found nothing. Melanin is a
pigment that plays a crucial role in the development of the eyes, skin, and hair.
There are several syndromes that include oculocutaneous albinism, intellectual
disability and other phenotypic presentations. Some of the examples have been
presented in . SPAG9 has been associated with functional clusters known to be
associated with neurodevelopmental disorders (NDDs) including chromatin
organization, nervous system development and revealed co-expression/ interaction
with other known candidate genes. Thus, suggesting a stronger influence in the
etiology of NDDs ( ).
Similarly, ) present an abstract describing a Colombian family with 9 affected
individual’s revealed homozygous variant (p.Tyr914Ter) in the SPAG9 with features such as severe intellectual disability (ID), speech delayed, gait
disturbance, cerebellar syndrome, cataracts, strabismus, seizures and eyelid ptosis.
Brain MRI revealed brain atrophy, hippocampal malrotation, thinning of the corpus
callosum and white matter hyperintensities (Acosta-Baena et al .,
2021). However, their patients do not have oculocutaneous albinism. These findings
support the data in the present study that mutated SPAG9 is a
potential candidate gene for the novel syndrome. The previously reported patients’
phenotypes clearly overlap. The OCA phenotypes not seen in the patients reported by
Acosta-Baena et al . (2021), could be related to a change in the
mutant domain (p.Tyr914Ter) as compared to ours (p.Phe301Leufs*2) or other
regulatory effects caused by the difference in the position of the identified
mutation. The SPAG9 gene has been associated with oculocutaneous albinism in
homozygous mutant mice models (http://www.informatics.jax.org/allele/MGI:4442906),
which also support our current study. The MGI revealed phenotypes such as diluted
coat color, absent skin pigmentation, and decreased activated sperm motility in the
homozygous knockout mice models
(https://www.informatics.jax.org/marker/MGI:1918084). Similarly, SPAG9 (MGI: 1918084) showed expressing in different mice organs
such as nervous system, visual system, and reproductive system. We clearly observe
overlapping of the phenotypes as shown in the knockout mice and humans. Our study
suggests SPAG9 gene as a strong candidate for oculocutaneous
albinism, developmental delay, cataract and coarse facial features due to loss of
function variant (LOF), however further evidences in humans are needed. Network biology has emerged as a promising approach to identify drug targets in
complex genetic disorders, which could be applicable to understanding the molecular
interactions involving SPAG9 in the novel syndrome identified
( ).
Similarly, the use of molecular and cellular biomarkers has been pivotal in
advancing our understanding of developmental toxicology, providing a framework that
could be adapted to explore the pathogenic mechanisms involving SPAG9 ( ). In conclusion, to our knowledge, this is the first time to define the phenotypic
spectrum of SPAG9 -associated with coarse facial features, albinism,
cataract, skeletal abnormalities, and developmental delay. We provide evidence that
biallelic LOF-variants in the SPAG9 gene might lead to a novel
syndrome in humans 

