Genetics and genomic characterization of Congenital Upper Limb Anomalies

Congenital upper limb anomalies (CULA) are rare developmental disorders arising
in approximately 1 in 500, CULA associated with a syndrome only arise in 0.5
out of 10.000 live births. There is a broad range of anomalies varying from
extra simple extra digits to complex syndromal hand deformities. Since 1976
CULA are described using the classification by Swanson adopted by the IFSSH.
Due to numerous genetic studies an increased comprehension of the molecular
basis of conditions with primary or secondary limb involvement had been
derived. These progressions have reflected in a new classification of limb
anomalies in 2009 by Oberg, Manske and Tonkin. This classification better
describies the embryonic correlation of these individual anomalies, but also
has been revised twice since publication.
Although many genes were identified in the past, the genotype-phenotype
relation is still often poorly understood as illustrated by GLI3 gene
mutations. GLI3 gene mutations can cause a variety of hand malformations in a
range from isolated polydactyly to Pallister Hall syndrome and Greig*s
Cephalopolysyndactyly. Moreover, in many defined syndromes like Greig*s
Cephalopolysyndactyly there is a wide variety in both phenotype and underlying
genotype. Common hypothesis for these variations are polygenetic alterations
hindering strict correlation between phenotype and the molecular or genetic
basis. Wider genetic testing and improved phenotypic registration could improve
understanding and thereby genetic counseling and patient care.
Researchers have shown that with different genetic research techniques more
mutations can be found but research for genetic defects is time and money
consuming with the standard techniques. The introduction of Next Generation
Sequencing (NGS) supplies us with reliable and quick results. This has
previously been shown with the identification of a mutation of the IL11RA gene,
causing craniosynostosis, by using NGS and simultaneously using more
conventional techniques for validation. Interpretation of the data obtained
through NGS requires a database with control samples to quickly filter and
eliminate the common, non-pathogenic genetic variations that occur in the
normal healthy population. The department of Bioinformatics has a unique
database containing more than 180 genomes that can serve as control samples.
The use of a transitional research center enables analysis and control
capabilities of both genetic and clinical data that scale to millions of
patients and hundreds of thousands of whole genome sequences. By collecting
detailed clinical data of the 2500 CULA patients of the Sophia*s Children*s
hospital and the data from the standard DNA tests performed by the Clinical
Genetics department cases fitted for whole genome sequencing can be selected
based on syndrome, malformation, location of the malformation, patient history
or previously detected genetic anomalies and any combination of the before
mentioned. When the first whole genome sequences are included the database can
also help identify like-cases fitted for sequencing.
Most centers lack a sufficient amount of patients for genetic research. The
Sophia Children*s Hospital is the major hand and upper limb surgery center in
the Netherlands and therefore rare congenital upper limb anomalies disorders
are centered here. Due to this, a database of over 2500 congenital upper limb
anomaly patients is available. Collaborations with the hand and upper limb
surgery center in Hamburg possibly extends the amount of includible patients
even further.
Drawing upon our database of over 2500 congenital upper limb anomalies
malformation patients, a pilot study will be conducted, comparing the costs of
conventional testing (about 750 euros per gene, and often not establishing a
genetic cause since only previously discovered mutations are tested) to whole
genome sequencing. Currently, the cost for a full genome is comparable to the
cost of several (five) single-gene tests, and provides the added ability to
identify novel causal variants. Futhermore having the knowledge about the whole
genome will save costs for insurance companies by the fact that certain future
tests or searches for variations will only involve a digital look-up without
any additional laboratory tests.

Primary Objectives:
1. Determine novel pathogenic gene mutations in families with congenital upper
limb anomalies by whole genome sequencing;
2. Determine the contribution of these genes in embryonic development,
including proliferation and cell death.

Secondary Objectives:
1. Evaluate the significance of whole genome sequencing in determining risk
genes in syndromal or hereditary congentinal upper limb abnormalities
2. Understand the heredity and the consequences of the new found mutations to
ameliorate counselling

There is no set age for patients with congenital upper limb anomalies to be
operated on, while the indication for operation depends on the function deficit
experienced by the patient or their parents and the moment of referral to the
outpatient clinic.
According to standard protocol, the parents receive a questionnaire prior to
their first visit about the pregnancy, family history, growth and development.
Standard physical examination includes full body inspection and evaluation of
all extremities. Both X-ray and photographic recordings are made of the
affected limb(s) and the healthy contralateral limb(s). Patients are referred
to the department of Clinical Genetics when a hereditary condition is
suspected. Patients will be asked to bring pictures of family members with
(upper limb)anomalies to the outpatient clinic of the Clinical Genetics
department. The patient and the photos will be analysed for the presence of
dysmorphisms. DNA analysis is performed when indicated, blood required in this
stage will be drawn during surgery. All data generated by standard protocol
will be included in an OpenClinica database compatible with the Translational
Research Center. Genomic data will be stored in the Huvariome database.
Patients for the first phase of whole genome sequencing are asked to join this
study when there is a suspicion of a hereditary anomaly but no known mutation
was found in standard DNA testing. When consent is given, family members with
CULA*s will be approached for inclusion in the database as well. The Department
of Clinical Genetics is involved for family counseling during the course of
this study.
All samples will be will be sent to the United States where whole genome
sequencing will be performed by Complete Genomics (Mountain View, CA, USA). DNA
profiles will be made. Associations between these profiles and the profiles of
150 normal genomes will be made. A number of risk genes will be evaluated for
function and pathogenicity and a final selection will be made by the department
of Bioinformatics of the Erasmus MC. The department of Bioinformatics has a
METC approval for conducting whole genome sequence analysis including
databasing of the results for re-use(MEC 2011- 253). As control the new found
mutation will be tested in affected and unaffected family members by
resequencing the gene. By cross-reference with our database incidences of the
new mutations can be estimated.
The anonymised genomic data derived from control individuals and patients will
be stored and therefore can be used for future analysis of additional CULA
patients.

The burden and risks are practically none. In phase 1 the blood used for whole
genome sequencing has already been collected during surgery for the regular DNA
testing. Sometimes more DNA will be necessary or family members will be asked
for blood in phase 2. Patients will be asked to complete a questionnaire and a
routine elaborate physical examination will be done at the outpatient
department.
The whole human genome will be mapped using whole genome sequencing. This will
offer lots of information about an individual. In this research project we will
focus on disease related genetic alterations. Since knowledge of genetic
alterations is expanding rapidly, our data cannot be checked for all new
discovered mutations. To prevent incomplete statements no statements will be
done about other genetic diseases.
An extensive look at all possible causative genetic alterations causing
congenital upper limb anomalies will be made. The patient will probably find
out what causes his or her congenital upper limb anomalies and more will be
known about its heredity. Counseling will improve for both the parents
regarding risks for a future pregnancy and for the patient regarding risk to
pass the condition on. Moreover, an enormous amount of knowledge concerning the
genetic background, embryology and development of congenital upper limb
anomalies will be gained. Future patients will benefit from this enhanced
knowledge in improved counseling and in future in personalized medicine.
Furthermore, the anonymized genome data will be stored and can be used for
future analysis of additional patient data.