Med Clin (Barc).

2015;145(4):147–152

www.elsevier.es/medicinaclinica

Original article

Algorithm for the molecular analysis of Bardet–Biedl syndrome

in Spain夽
Sheila Castro-Sánchez, María Álvarez-Satta, Inés Pereiro, M. Teresa Piñeiro-Gallego, Diana Valverde ∗
Departamento de Bioquímica, Genética e Inmunología, Facultad de Biología, Universidad de Vigo, Vigo, Pontevedra, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Background and objective: Bardet–Biedl syndrome (BBS) is a multisystemic genetic disorder, which is not
Received 30 January 2014 widespread among the Caucasian population, characterized by a highly variable phenotype and great
Accepted 8 May 2014 genetic heterogeneity. BBS belongs to a group of diseases called ciliopathies, caused by defects in the
Available online 11 February 2016
structure and/or function of cilia. Due to the diagnostic complexity of the syndrome, the objective of this
study was to analyze our whole group of patients in order to create an algorithm to facilitate the routine
Keywords: molecular diagnosis of BBS. We also calculated several epidemiological parameters in our cohort.
Ciliopathies
Patients and method: We analyzed 116 BBS patients belonging to 89 families from the whole Spanish
Prevalence
Bardet–Biedl syndrome
geography. All probands fulfilled diagnosis criteria established for BBS. For this, we used: genotyping
Diagnosis algorithm microarray, direct sequencing and homozygosis mapping (in consanguineous families).
Results: By means of the different approaches, it was possible to diagnose 47% of families (21% by genotyp-
ing microarray, 18% by direct sequencing of predominant BBS genes, and 8% by homozygosis mapping).
With regard to epidemiological data, a prevalence value of 1:407,000 was obtained for BBS in Spain, and
a sex ratio of 1.4:1 (men:women).
Conclusions: The proposed algorithm, based on the analysis of predominant BBS genes combined with
homozygosis mapping, allowed us to confirm the molecular diagnosis in a significant percentage of
families with clinically suspected BBS. This diagnostic algorithm will be useful for the improvement of
the efficiency of molecular analysis in BBS.
© 2014 Elsevier España, S.L.U. All rights reserved.

Algoritmo para el estudio molecular del síndrome de Bardet-Biedl en España

r e s u m e n

Palabras clave: Fundamento y objetivo: El síndrome de Bardet-Biedl (SBB) es una enfermedad genética multisistémica
Ciliopatías poco frecuente en población caucásica, caracterizada por una pronunciada variabilidad fenotípica y una
Prevalencia gran heterogeneidad genética. Pertenece al grupo de las ciliopatías, causadas por defectos en la estruc-
Síndrome de Bardet-Biedl
tura y/o función ciliar. Dada la gran complejidad diagnóstica del síndrome, el objetivo de este estudio ha
Algoritmo diagnóstico
sido analizar el conjunto global de afectados recogidos para elaborar un algoritmo que facilite el diagnós-
tico molecular rutinario del SBB, así como calcular algunos parámetros epidemiológicos para población
española.
Pacientes y método: Se han analizado 116 afectados de SBB pertenecientes a 89 familias procedentes de
toda la geografía española. Todos los probandos cumplían los criterios diagnósticos establecidos para
el SBB. Para ello, se utilizaron las siguientes técnicas: microchip de genotipado, secuenciación directa y
microchip de homocigosidad para familias consanguíneas.
Resultados: Ha sido posible diagnosticar al 47% de las familias (21% mediante el microchip de genotipado,
18% mediante secuenciación directa de genes BBS predominantes y 8% mediante el mapeo de regiones
homocigotas). En cuanto a los datos epidemiológicos, se obtuvo un valor de prevalencia del SBB en España
de 1:407.000, así como una razón por sexos de 1,4:1 (varones:mujeres).

夽 Please cite this article as: Castro-Sánchez S, Álvarez-Satta M, Pereiro I, Piñeiro-Gallego MT, Valverde D. Algoritmo para el estudio molecular del síndrome de Bardet-Biedl
en España. Med Clin (Barc). 2015;145:147–152.
∗ Corresponding author.
E-mail address: dianaval@uvigo.es (D. Valverde).

2387-0206/© 2014 Elsevier España, S.L.U. All rights reserved.

148 S. Castro-Sánchez et al. / Med Clin (Barc). 2015;145(4):147–152
Conclusiones: El algoritmo propuesto, basado en el análisis de genes BBS predominantes combinado con
estudios de homocigosidad, ha permitido confirmar el diagnóstico molecular en un porcentaje significa-
tivo de familias con sospecha clínica de SBB. Este algoritmo diagnóstico permitirá optimizar el análisis
molecular del SBB.
Introduction
Bardet–Biedl syndrome (BBS, MIM # 209900) is a rare genetic
disease, infrequent in Caucasian population with an estimated
prevalence, in relation to live births, of 1:125,000–1:160,000 in
Europe1,2 and 1:100,000–1:140,000 in North America.3 However,
there are some isolated populations with much higher figures,
such as the Faroe Islands (Denmark), with a value of 1:3700 live
births,4 that would be explained as a consanguinity problem due
to its low population, and potential founder effect, given the geo-
graphic isolation. As a matter of fact, one of the highest BBS
prevalence in Europe so far has been reported in Danish population:
1:59,000 live births.5 In addition, BBS shows different prevalence
rates depending on sex, being slightly higher in males, with a 1.3:1
ratio.6
BBS is a multisystemic disease with a significant intrafamilial
and interfamilial phenotypic variability.7 As a result, the clin-
ical manifestations have been categorized in terms of relative
prevalence, thus establishing a primary or cardinal and a sec-
ondary number of manifestations6 (Table 1). Currently 6 primary
characters have been considered: retinal dystrophy, obesity, poly-
dactyly, hypogonadism, kidney anomalies and dealyed cognitive
development.6,8 Retinal dystrophy is the most common mani-
festation of BBS, and is presented as an early and progressive
degeneration of retinal photoreceptors, starting as a night blind-
ness, and then developing photophobia and loss of central and
color vision.8 Central obesity and posaxial polydactyly (although
cases of mesoaxial polydactyly have also been reported9 ) are 2
of the most frequent manifestations.8 BBS patients may also have
various genital malformations, mainly hypogonadism as well as
various renal anomalies, the most common being tubular cys-
tic disease and various anatomical malformations.8 BBS patients
may also present delayed cognitive development, often accompa-
nied by behavioral disorders, learning difficulties or psycomotor
problems.8 Among the various secondary manifestations, type
2 diabetes mellitus, dental and palatal malformations, brachy-
dactyly/syndactyly and anosmia have been reported.8 The presence
of four primary manifestations or 3 primary and 2 secondary
Table 1
Primary and secondary clinical characteristics present in Bardet–Biedl.
Primary characteristics
Retinal dystrophy
Obesity
Polydactyly
Hypogonadism
Renal anomalies
Delayed cognitive development
Secondary characteristics
Diabetes mellitus
Dental and palatal malformations
Brachydactyly/syndactyly
Anosmia
Speech delay
Psychomotor retardation
Behavioral changes
Craniofacial anomalies
Cardiovascular problems
© 2014 Elsevier España, S.L.U. Todos los derechos reservados.
manifestations is required to confirm the clinical diagnosis of
BBS.6
BBS belongs to a group of disorders known as ciliopathies,
which share a common etiology: defects in the cilia structure and
cilia dysfunction.10 This group includes other syndromes, such as
Alström, Meckel-Gruber and Joubert, all being multisystemic dis-
orders with overlapping phenotypes,11 making accurate and early
diagnosis to be very difficult (Figure 1). In addition, delayed and pro-
gressive onset of most BBS manifestations, along with the existence
of genes common to various ciliopathies, make an early diagnosis
to be even more difficult.8
BBS is usually an autosomal recessive disease, with 18 genes
having been reported (BBS1–18) to be involved in its development
so far.10,12–14 This would explain about 75–80% of patients with
clinically suspected BBS,8,15 which shows the great genetic het-
erogeneity associated. Furthermore, 2 predominant mutations: p.
(Met390Arg) in BBS1 gene and p. (Cys91Leufs * 4) in BBS10 gene
have been described in European populations.16,17
Epidemiological data on BBS are very limited in Spain due
to its high diagnostic complexity and the percentage of under-
diagnosed or misdiagnosed cases. Therefore, this study has been
aimed at analyzing the global data registered from patients with
BBS by our group since 1990, and creating a molecular diag-
nostic algorithm to facilitate routine study of this syndrome,
often tedious and complex. It has also been intended to calculate
some epidemiological parameters for the first time in a Spanish
cohort.
Patients
Since 1990, 116 samples have been registered from BBS patients
belonging to 89 families selected from various hospitals and med-
ical centers throughout Spain. Seven of them are of Arab or Indian
origin, and 15 families have been proved to show various degrees of
consanguinity. All probands meet the BBS diagnostic criteria estab-
lished by Beales et al.6
The study complies with the principles of the Declaration of
Helsinki and was approved by the Ethics Committee on Clinical
Research of Galicia. Also, the informed consent of all partici-
pants, or their legal representatives, was obtained after having
explained in detail the procedures that would be performed in this
study.
Methods
In previous studies18–20 (and unpublished observations) muta-
tional load of a high percentage of families has been characterized
following different strategies, which are summarized below: the
89 families were analyzed by microchip genotyping18 (and unpub-
lished observations) that contains the most frequent sequence
variations found in various BBS genes, among others. After this
first approach, patients with only one or no mutation detected,
were studied by direct sequencing of the BBS genes most involved
in this syndrome (BBS1, BBS10 and BBS12)20 and references con-
tained. To this purpose, we proceeded to the extraction of DNA
from peripheral blood of patients using FlexiGene® DNA kit 250
(Qiagen, Hilden, Germany). Next, the coding regions and flank-
ing intronic sequences of BBS1, BBS10 and BBS12 genes were
 S. Castro-Sánchez et al. / Med Clin (Barc). 2015;145(4):147–152 149

b Skeletal defects
a
BBS

BBS1 BBS8 NPHP

BBS3 BBS9 GLIS2 ECV
BBS5 BBS10 NEK8
BBS7 BBS12

NPHP2
NPHP4
MKS1 BBS6 NPHP5
BBS2 BBS4 NPHP6
JATD
MKS3 FTM
OFD1
MKS2
NPHP3 NPHP1
IQCB1
NPHP JBTS
CC2D2A PKD
SLSN BBS
MKS SLSN
ALMS MKS
AH1
ARL120
CXORF5

JBTS Renal cysts Retinal dystrophy

Fig. 1. (A) Genetic overlap between ciliopathies. It shows how a single gene may be responsible for the development of various syndromes. (B) Phenotypic overlap between
ciliopathies. Syndromes are classified in relation to the presence or absence of 3 common phenotypic characteristics. Modified from Quinlan et al.26 ALMS, Alström syndrome;
BBS, Bardet–Biedl syndrome; EVC, Ellis–van Creveld syndrome; JATD, Jeune syndrome; JBTS, Joubert syndrome; MKS, Meckel–Gruber syndrome; NPHP, Nephronophthisis;
OFD1, Orofaciodigital syndrome type 1; PKD, polycystic kidney disease; SLSN, Senior–Loken syndrome.

amplified by polymerase chain reaction with primers described in
the medical literature.16,17,21 Finally, the purified DNA products
were sequenced using BigDye® Terminator v1.1 Cycle Sequencing
Kit (Life Technologies, Foster City, CA, USA) and were resolved in
an ABI PRISM® 3130 sequencer (Life Technologies, Foster City, CA,
USA). Those deletions or duplications identified in heterozygosity
were studied by cloning using pGEM® -T Vector System I (Promega,
Madison, WI, USA).
Moreover, 13 of the consanguineous families were studied using
the GeneChip® Human Mapping 50K (Affymetrix, Santa Clara, CA,
USA) with the purpose of detecting homozygous chromosomal
regions, candidate for harboring mutations in BBS genes or new
loci potentially involved in BBS19 (and unpublished observations).
Results
The different approaches used in this study have confirmed the
clinical diagnosis, i.e. 2 pathogenic variants have been reported,
registered from 42 of the families, representing 47% of the total.
Therefore, 53% of them have not been confirmed yet at a molecular
level.
Breaking down the data according to the technique used, the
microchip genotyping has allowed the diagnosis of 21% families (19
in 89 families), direct sequencing of predominant BBS genes in 18%
(16 in 89 families, including 5 families with a single mutation iden-
tified by microchip), and the remaining 8% (7 in 89 families) were
diagnosed using the homozygosity mapping (Table 2). As for the
studies on homozygosity, it is necessary to point out that although
the percentage obtained on the total number of families is not high,
it should be noted that in over 50% of the consanguineous families
studied by this technique (7 of 13) the two mutated alleles have
been identified.
Regarding the epidemiological data, it has been possible to cal-
culate for the first time an estimated BBS prevalence in Spain: one
person affected per 407,000 individuals. This has been estimated
considering the total number of individuals affected by this condi-
tion and registered by our group and the latest Spanish population
data published by the Spanish National Institute of Statistics. As for
the cohort distribution by sex, BBS affects more males than females,
with a 1.4:1 ratio.
From the above data, an algorithm for diagnosing BBS was devel-
oped to facilitate molecular study and avoid a laborious analysis
per gene, given the genetic heterogeneity associated and the vir-
tual absence of predominant mutations in different populations
(Figure 2). Thus, the proposed algorithm is performed as follows:
(a) assessment of the 2 predominant mutations described for BBS;
(b) direct sequencing of the predominant BBS genes (BBS1, BBS10
and BBS12); (c) in the case of consanguineous families, detec-
ting candidate homozygous regions by homozygosity microchip
and sequencing the BBS genes located therein. If none of the
above approaches is successful, and in the case of families with
no proven consanguinity, further strategies would be applied, such
as the whole exome sequencing or searching and studying new
candidate genes, mainly related to ciliary activity. These new can-
didates might be responsible for BBS in some of the families
registered.
Table 2
Percentage of families diagnosed of Bardet–Biedl syndrome with the techniques
used in this study compared to the total families registered (89).
Method of diagnosis Percentage of families
with mutation
Microchip genotyping 21
Direct sequencing of predominant 18
genes (BBS6, BBS10, BBS12)
Homozygosity microchipa 8
Undiagnosed 53
a
Seven of the 13 consanguineous families studied have been diagnosed with this
technique.
150 S. Castro-Sánchez et al. / Med Clin (Barc). 2015;145(4):147–152

Analyze p.(Met390Arg) (BBS1) and p.(Cys91Leufs*4) (BBS10)

2 alleles mutated 1 allele mutated No allele mutated

End of study Sequencing the remaining

genes (BBS1 o BBS10)

Sequencing the
2nd allele mutated 2nd allele non mutated predominant BBS genes
(BBS1, BBS10 y BBS12)

End of study

None or 1 allele mutated 2 alleles mutated

End of study

No consanguineous families Consanguineous families

Homozigosity
microchip

Homozygotes regions than Homozygotes regions

Ultrasequencing do not include BBS genes include BBS genes
studies (NGS)
and/or search and
squencing of new
candidate genes Sequencing BBS genes

None or 1 allele mutated 2 alleles mutated

End of study

Fig. 2. Algorithm for the molecular analysis of Spanish patients with Bardet–Biedl syndrome. NGS, next generation sequencing (“new massive sequencing platforms”).

Discussion The use of microchip genotyping provides significant advan-

This study is relevant for BBS in Spain, where little data is avail-
able on this syndrome.18–20 Thus, it has been possible to establish
the first prevalence data in Spain, with a 1:407,000 ratio, signif-
icantly lower than in other European populations.1,2,4 However,
potential misdiagnosis should be considered, as well as the per-
centage of cases underdiagnosed, mainly due to the overlapping
phenotypes among ciliopathies, which complicates the clinical
diagnosis of this group of disorders. A sex-related ratio was also
obtained, 1.4:1. This data is consistent with the study by Beales
et al.6 (and previous references mentioned therein), but differs from
Green et al.,22 who observed a slight excess of women.
Given the high genetic heterogeneity associated with BBS, it is
essential to optimize resources in molecular diagnosing, mainly
those aimed at reducing time and cost, and providing an accurate
and early diagnosis. Based on our previous experience characteriz-
ing the mutational load of families using the microchip genotyping,
direct sequencing of predominant BBS genes and the homozygosity
studies,18–20 a working algorithm for the molecular analysis of this
syndrome has been designed (Figure 2).
tages in genetic analysis of heterogeneous diseases such as BBS, and,
in fact, has helped confirm the diagnosis of 19 families. However,
most changes detected are related to the 2 predominant muta-
tions in this syndrome, mainly the p. (Met390Arg) mutation in
BBS1. This might be due to the high percentage of private muta-
tions identified in this syndrome (present in one family),15,23 and
subsequently they have not been incorporated into the microchip.
Therefore, this approach could be replaced by direct sequencing of
the 2 predominant mutations, thereby reducing analysis time and
cost.
Taking into account the References’ data,15–17 as well as the
results of the microchip genotyping, BBS1 and BBS10 genes have
been selected as a priority for our study cohort. BBS12 gene
analysis has also been considered, given the increasing rele-
vance acquired in recent years.15 The results obtained after direct
sequencing of these 3 genes confirmed their importance to search
mutations responsible for the disease allowing diagnosis to 16
families. Moreover, the structure of the BBS10 (2 coding exons)
and BBS12 (one coding exon) genes makes them ideal for quick
analysis.
 S. Castro-Sánchez et al. / Med Clin (Barc). 2015;145(4):147–152
In addition, 13 of the 15 families with different consanguinity
degree were analyzed using homozygosity microchip.19 Through
this approach it has been possible to diagnose 7 of the 13 families.
This lets us confirm that this is a useful technique to detect homozy-
gous regions matching the chromosomal location of BBS genes, as
well as other candidate genes responsible for the development of
BBS.
Therefore, the analysis of mutations and predominant BBS genes
is a good diagnostic strategy in our cohort, which combined with
studies on homozygosity, can confirm the diagnosis in a signifi-
cant percentage of families with clinically suspected BBS. However,
approximately 50% of the families registered remain to be uncon-
firmed at a molecular level. But we should take into account that
mutations might exist in the unanalyzed BBS genes in this study
and that genetic heterogeneity in Spain is very high compared
to other European populations,5,15 where a number of predomi-
nant mutations have been reported and this facilitates their genetic
study.
In these families where homozygous mutations have not been
reported or with no homozygous sequences consistent with BBS
genes, the next step would be the next generation sequencing
(NGS, “new massive sequencing platforms”). With these tech-
nologies it is possible to obtain thousands of DNA sequences
rapidly and detect all types of genomic variation in a single
experiment, so it is a very useful technology for the molecular
diagnosis of genetically heterogeneous disorders.24,25 However,
these techniques have some drawbacks, such as its current high
cost and difficulty managing the vast amount of data generated,
which requires a certain knowledge on bioinformatics for proper
interpretation.24 Furthermore, to perform such analyses some DNA
quality and quantity is required, so analyzing all the collected
samples is not always possible.24 Another important factor is cov-
erage of genes of interest, as it varies depending on the genes.
This coverage might be 10% only, so there would be a high proba-
bility of not detecting potential pathogenic changes in uncovered
sequences.24
Identifying new candidate genes is one more advantage of these
ultrasequencing techniques, since it would allow better under-
standing of the mechanisms involved in the disease.24,25 In the near
future these techniques will be more viable, since their cost will
be lower, and an easy-to-use software will be available for inter-
preting data.24,25 Therefore, despite the good expectations on these
NGS techniques, currently it is possible to diagnose approximately
half of the families with clinically suspected BBS using classical
approaches such as direct sequencing of predominant genes or find-
ing homozygous regions in consanguineous families in a simpler
and cheaper manner.
Authors
Sheila Castro-Sánchez and María Álvarez-Satta have also con-
tributed in preparing the manuscript.
Funding
This project has been funded by the FIS PI12/01853 project
granted by the Spanish Ministry of Economy and Competitiveness.
María Álvarez-Satta is a beneficiary of a FPU grant, granted by
the Ministry of Education, Culture and Sports, and Sheila Castro-
Sanchez is the recipient of a predoctoral contract by Xunta de
Galicia.
Conflict of interests
The authors declare no conflict of interests.
151
Acknowledgments
We would like to acknowledge all the families who have partic-
ipated in this study and the various hospitals and medical centers
that have provided samples. We would also like to acknowl-
edge the Spanish Wolfram, Bardet-Biedl and Alström syndromes
Register (REWBA), the European Registry for Rare Diseases for Wol-
fram, Alström, Bardet-Biedl syndromes and other rare diabetes
syndromes (EURO-WABB Registry) and the Program to Support
Biomedical Capacity (BIOCAPS).
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